Degradation of polycyclic aromatic hydrocarbons to render them available for biodegradation

ABSTRACT

A method for the degradation of polycyclic aromatic compounds is disclosed that involves dissolving ozone in a bipolar solvent comprising a non-polar solvent in which is of sufficiently non-polar character to solubilized the polycyclic aromatic compounds, and a polar-water-compatible solvent which is fully miscible with the non-polar solvent to form a single phase with the non-polar solvent. The bipolar solvent with dissolved ozone is contacted with the polycyclic aromatic compounds to solubilize the polycyclic aromatic compounds and react the dissolved polycyclic aromatic compounds with the ozone to degrade the dissolved polycyclic aromatic compounds to oxygenated intermediates. The bipolar solvent is then mixed with sufficient water to form separate non-polar and polar phases, the non-polar phase comprising the non-polar solvent and the polar phase comprising the non-polar solvent and the oxygenated intermediates. The polar phase is then diluted and incubated with bacteria to biodegrade the oxygenated intermediates.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/129,354, entitled DEGRADATION OF POLYCYCLIC AROMATICHYDROCARBONS TO RENDER THEM AVAILABLE FOR BIODEGRADATION, which claimspriority to the International Application under the PCT number:PCT/US00/30599, international filing date 6 Nov. 2000, which claimspriority to U.S. Provisional Patent Applications 60/164,070 and60/164,071, both filed 5 Nov. 1999. All such are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

(Not applicable)

FIELD OF THE INVENTION

This invention relates to the chemical degradation of polycyclicaromatic hydrocarbons to render them available or biodegradation.

BACKGROUND OF THE INVENTION

Polycyclic aromatic hydrocarbons (PAHs) are a group of aromaticcompounds containing two or more fused benzenoid rings in linear,angular, or cluster structure. They are ubiquitous compounds that areformed naturally during thermal geologic reactions, plant fossilization,and bacterial reactions, or formed anthropogenically during mineralproduction, combustion of fossil fuels in heat and power generation,refuse burning, coke oven, pyrolysis, and forest and agricultural fires.The major sources of PAHs are crude oil, coal, and oil shale.Hydrophobic, recalcitrant, and bio-accumulating, PAHs adsorb strongly tosuspended particulates and biota, and accumulate in soil and sediment,resulting in serious soil contamination problems. Health concerns ofPAHs arise from their toxicity, mutagenicity, and carcinogenicity. Ofthe 16 PAHs listed by the US EPA as priority pollutants due to theirtoxic and mutagenic nature, six are also known to be carcinogens. PAHsare known as active carcinogens. Their presence is an indicator ofindustrial pollution, and they are widely distributed in contaminatedenvironments, particularly prevalent in burnt organic matter, air, andcontaminated soil.

Both biological and chemical techniques have been used for theremediation of PAHs, although bioremediation is generally found toderive cost and technical advantages. While low-molecular-weight PAHsare susceptible to biodegradation, high-molecular-weight (HMW) PAHs thatare highly mutagenic and carcinogenic remain recalcitrant. Therefractory nature of HMW PAHs is partly attributed to their low aqueoussolubility and bioavailability, with their degradation rates possiblylimited by dissolution or desorption.

Chemical oxidation using electrophile O₃ has been seen as a treatmentfor PAH compounds in the aqueous phase or in solution. PAH compounds,such as benzo[a]pyrene, in organic solvents or in various aqueoussolvents have been treated with ozone to form oxygenated products.However, these are limited in their utility for remediation because theyrequired that the PAH compounds be in solution. Since the solubility ofmany PAH compounds is water is low, such solution treatment with ozoneis limited as it treats only the more soluble compounds.

PAH compounds may be more soluble is certain organic solvents such asethylene or methylene chloride, but these solvents in themselves presentenvironmental problems and are accordingly undesirable for environmentalremediation applications. In addition, the reaction products of theozone and PAHs are often insoluble in these organic solvents, causinginsoluble solid precipitates. Addition of water to these systems tosolubilize the intermediates creates a multiphase system that isdifficult to handle.

One of the more severe environmental problems involving PAHs is derivedfrom oil spills. Oil spills are known for causing long-term and severedamage to environment. Biodegradation, volatilization, oxidation, andphotochemical reactions alter a limited amount of the oil; the remainderof the oil is dissolved into water, and/or dispersed into soils. Manyhigh molecular weight and hydrophobic compounds such as polycyclicaromatic hydrocarbons (PAHs) and aromatic sulfur compounds areaccumulated due to their toxicity and poor water solubility, thusinaccessible to microbes and even to chemical oxidant such as O₃ in theaqueous phase.

Petroleum released into environments have been remediated with a widerange of chemical, physical, and biological processes. Differentfractions of oil spills can transform or degrade through evaporation,plant uptake, and dissolution into water, adsorption by soil matrix,photo-oxidation, and biodegradation. Among all the attenuationphenomena, biodegradation is the primary mechanism for contaminantdestruction. Biodegradation of oil in terrestrial and aquaticenvironments is currently the most widely accepted option forpetroleum-contaminated sites. The biological degradation of oil can betaken place in aerobic or anaerobic environments, although aerobicbiological oxidation is regarded as more efficient. In low oxygenconditions, such as in a oil-polluted ground water environment, thebiotransformation of hydrocarbons can also occur when the nitrate,sulfate, carbon dioxide, and ferric iron were utilized as alternateelectron acceptors. However, petroleum degradation under anaerobiccondition is generally considered to be difficult due to the limitedgrowth substrate, electron acceptors, and enzymatic activities.Accordingly, aerobic biological oxidation of hydrocarbons is consideredto be the major biodegradation processes.

The preferential biodegradability of fractions in the crude oil spillhas been reported as the n-alkanes>branch alkanes>cyclicalkanes>aromatics. In addition, volatile aromatic fractions (i.e.benzene and toluene) of oil have short residence times in theenvironment. Having a low preference for biodegradability with lowbioavailability, low enzymatic activity, and a low volatility,high-molecular weight and hydrophobic compounds of petroleum accumulate.For example, the cyclic-alkanes and polycyclic aromatic hydrocarbonsfrom oil spills will stay in the environment for a long period of time.Especially, PAHs are relatively stable and diagnostic constituents ofpetroleum. The biodegradability of polycyclic aromatic compounds arelimited by their toxicity and water solubility because of most of thebiodegradation occurring in the water or water-oil interface. Thus, theaccumulation, persistence, and mobility/leaching potential of toxic PAHseven with effective bioremediation are still the major health andenvironmental concerns. In other words, although bioremediation can be acost-effective method to remove considerable amounts of oil spills, thecontaminant concentration cannot be completely eliminated because ofthese persistent PAH compounds.

Ozone for the oxidations of PAH compounds in aqueous solutions has beenfound to effective for those compounds in solution, but is not effectivefor these compounds that are essentially insoluble. Wastewatercontaining recalcitrant organic compounds has been successfully treatedwith ozone. Many studies on degradation of PAHs by ozone proven canimprove the solubility and decrease the toxicity of PAH compounds.

In summary, the prior-art shows (1) treatment of water soluble PAHcompounds with ozone in water, generally for waste water treatment, (2)treatment of PAH in non-polar solvents with ozone, sometimes inconjunction with a non-miscible solvent to form two phases. The mainproblem with these systems is that non-soluble PAH compounds are notavailable to water solution and escape reaction with ozone. The problemwith non-polar solvents is that such solvents are often toxic inthemselves, and introduce their own environmental problems. In addition,the non-polar solvents do not effectively dissolve the oxygenatedreaction products of ozone and the PAH compounds. Thus, these compoundscan precipitate from the solvent and are not removed.

OBJECTS OF THE INVENTION

It is, therefore, an object of the invention to provide a more effectivemethod for remediation of PAH pollutants.

Another object of the invention is to provide a method for the removalor degradation of PAH compounds that can successfully attack insolublePAH compounds in-situ.

Another object of the invention is to provide a method of thedegradation of PAH compounds the reduces the PAH compounds to readilydisposable or mineralized products.

Further objects of the invention will become evident in the descriptionbelow.

BRIEF SUMMARY OF THE INVENTION

The present invention involves the using an integrated approach foreffective treatment of PAHs, which involves chemical oxidation as apretreatment and biological treatment in the subsequent step. Thechemical oxidation is through reactions of O₃ and its concomitant OH.radical with recalcitrant PAH compounds, causing ring-cleavage andproducing hydroxylated intermediates such as aldehydes and acids thatare more soluble. The intermediates are thus rendered more amenable tofurther chemical or biological degradation in the aqueous solution.

An important feature of the present invention involves the use of abipolar solvent system, in conjunction with ozonation. The use of thebipolar solvents of the invention has been found to be more effectivethan the prior-art in treating and oxidizing high molecular weight andhydrophobic compounds such as polycyclic aromatic hydrocarbons (PAHs)and aromatic sulfur compounds that accumulated due to their toxicity andpoor water solubility. While these compounds have previously beeninaccessible to microbes and even to chemical oxidant such as O₃ in theaqueous phase, the practice of the present invention has allowed thesecompounds to be effectively treated.

The bipolar solvent system comprises (1) a non-polar hydrocarbonsolvent, such as heptane, and (2) a polar, hydrophilic solvent, such asacetic acid. The non-polar component enables high concentrations of PAHmolecules to be dissolved, while the polar hydrophilic solvent keeps thepolar intermediates and byproducts in solution. This bipolar solventsystem maintains effective exposure of all compounds to ozone throughoutthe course of reaction and prevents the formation of solid residues.Complete mineralization of PAHs, aromatic sulfur compounds and itsdaughter intermediates is possible by prolonged ozonation, or bybiodegradation following a shorter duration of ozonation pretreatment.

A suitable non-polar solvent is one that solubilizes the non-polar PAHcompounds. It should be immiscible in water, so that two phases will beformed upon mixture with water, and miscible with the polar solventselected. Suitable solvents include, but are not limited to fullysaturated hydrocarbons, such as liquid straight- or branch-chainhydrocarbons of 7 or more carbons, and halogenated hydrocarbons.Solvents, such as halogenated hydrocarbons that are toxic, are notpreferred, but are contemplated when their use is appropriate. Asuitable solvent is heptane.

The polar solvent has a sufficient hydrocarbon character such that it ismiscible in the non-polar solvent, but also be sufficiently polar to bemiscible in water such that it will partition into an aqueous polarphase with the polar compounds when the bipolar solvent system is mixedwith water. The polar solvent should also solubilize the polar compoundsthat are produced by the ozone reaction, as a function of the polarsolvent in the bipolar solvent is to retain the polar reaction productsin solution, and prevent their precipitation. Suitable polar solventsinclude organic acids such as acetic acid.

Ozone is dissolved in the bipolar solvent in sufficient quantity toreact with the PAH compounds. Accordingly, the bipolar solvent shouldhave sufficient ozone solubility to dissolve ozone in a reactive amount.The bipolar solvent stabilizes the ozone in solution to allow itsufficient time to react with the PAH compounds. For this reason, thebi-polar solvent constituents should be stable toward ozone, i.e., theydo not significantly react with the ozone before it reacts with the PAHcompounds. For this reason, alkane hydrocarbons, either straight orbranched, are preferred for the non-polar solvent as these are stabletoward ozone and have adequate ozone solubility.

To bring about bio-remediation, water is added to the reaction mixtureresulting in the formation of two distinct phases. The lighter uppernon-polar (heptane) phase contains any remaining parent PAHs, non-polarremnants of the PAHs, and little if any hydrophilic intermediates,whereas the heavier lower aqueous hydrophilic (acetic acid) solutionaccommodates a plethora of polar intermediates formed during ozonation.The amount of water is not critical, only sufficient need be added tocreate two-phases, which is only a small amount (usually about 5%). Theaqueous phase contains only small polar hydrocarbon remnants of the PAH,which are comparatively harmless, and may be disposed of or treated byknown methods. The non-polar phase may be treated to recycle thenon-polar solvent.

The polar phase is mainly the polar solvent with dissolvedintermediates. For bioremediation, the polar phase is separated from thenon-polar phase and diluted to a degree where it can support appropriatemicrobes. After dilution, the polar phase is bioreacted in the presenceof microbes, such as bacteria. The oxygenated intermediates aremetabolized to simpler compounds. In many systems, the intermediates maybe essentially mineralized (converted to non-organic compounds, such ascarbon dioxide and water) if desired. The intermediates laden solution(usually about 95% water) has a high biodegradability and a lowtoxicity. According, inoculation or other exposure of the solution withcommon microbes for bioreaction, such as E-coli, will reduce theintermediates to smaller molecular weight compounds that are readilydisposable.

Ozonation as a pretreatment for PAH deposits (such as oil spills) haspotential to eliminate the toxic portion of the deposit and provide morebioavailable and water-soluble degraded PAHs as well as biodegradablesaturated fractions for subsequent biological attenuation. The bipolarsolvent accommodates higher ozone concentration as well as being astable solvent for ozone. This system also provides the hydrophilicpolar solvent constituent that accommodates well the polar intermediatesproduced by the ozonation.

The steps of the method of the invention can be executed in any order,as long as the objective of solubilizing the PAHs achieved. The basicsteps include the combination of polar solvent, non-polar solvent,ozone, and contact with the PAH compounds. The PAH compounds may be in avariety of forms, such as dissolved species to the solid state. Anexample of an embodiment of the invention is shown in FIG. 24, whichshows (a) mixing of non-polar and polar organic solvent (b) to create amiscible phase bipolar solvent, (c) saturating the bipolar solvent withozone, (d) reacting PAHs with the ozone by adding PAH containingmaterials (crude oil) to the bipolar solvent, and (e) adding water toseparate the miscible phase into non-polar and polar phases. In FIG. 25is shown an alternate embodiment which shows, (a) dissolving PAHs (incrude oil) in a non-polar solvent, (b) adding a polar solvent to (c)form a single miscible phase with dissolved crude oil constituents, (d)introducing O₃ into the solution to react the PAH compounds, and (e)adding water to separate the miscible phase into a polar phase and anonpolar phase. The result, in any case, is a bipolar solvent containingdissolved oxygenated intermediates.

An application of the present invention involves the treatment ofspilled oil by ozone dissolved in a miscible non-polar solvent and polarsolvent system to break the aromatic rings of the polycyclic aromatichydrocarbons (PAH) to make them more adaptable to biodegradation bybacteria. As described above, the non-polar solvent serves to dissolvethe crude oil compounds and to carry off the non-polar substances. Thepolar solvent assists in carrying the ozonated intermediates into anaqueous phase where they are more available to bacterial attack. The PAHcompounds are thus reacted to form products that can be more easilybroken down to harmless product and metabolized by microbes. The presentinvention provides a method of the treatment of PAH compounds in oilspills that are toxic, solid and insoluble in water, which otherwisewould render these compounds difficult to remove and difficult forbacteria to metabolize. The ozone reacts with the PAH to open aromaticrings, making them more available as a food source, and increases oxygenfunctional groups which increase water solubility.

The ozone is dissolved in a solution of miscible non-polar and polarsolvents. The non-polar solvent dissolves the non-polar PAH compoundsand reaction products, making them available to attack by the ozone toform polar products, which are water-soluble. The non-polar solventdissolves the crude oil, i.e., hydrophobic and non-polar compounds,which renders the oil accessible to ozone treatment.

The ozone dissolved in the bipolar solvent reacts with PAH compoundsdissolved in the bipolar solvent, as well with the surface of theundissolved PAH compounds. Reaction at the solid PAH surface also servesto form more soluble compounds that can then be dissolved in thebi-polar solvent, thus degrading the solid surface. This increases thesurface subject to reaction, as well as releasing more soluble compoundsinto the solvent. Solid hydrocarbon residues usually contain materialsother than PAHs, such as mineral scale and wax deposits. While theseother materials are not attacked as well be ozone, removing andsolubilizing the PAH compounds can remove the physical integrity of thedeposit, allowing it to break up and be carried off in the solvent insmall particles. In summary, the bipolar solvent with dissolved ozonereacts PAH compounds dissolved in the bipolar solvent, with solid PAHsat the surface of solid residues, and with PAHs in free-floatingparticles freed from the solid surface.

The presence of the polar solvent allows the polar reaction productsthat are formed to be solubilized. Otherwise, these materials wouldprecipitate out of the non-polar solvent, forming undesired solidresidues. The presence of the polar solvent maintains the reactioncompounds in solution, which allows further breakdown by reaction withozone or biodegradation.

After contacting the ozone/non-polar/polar solvent system with thepetroleum residues, water is added to solvent system to form two phases.The first phase contains mostly the non-polar solvent and non-polar,hydrophobic constituents and products. The second phase contains mostlywater, the polar solvent and the polar products formed from reactionwith the ozone. The water increases the biodegradability of the polarmaterials by creating an aqueous phase.

The invention is applicable for treatment PAH deposits that occur inunderground hydrocarbon reservoirs, such as bitumen deposits and tarmats, as well as oil reservoirs, gas wells, and gas storage facilities.

For treatment of tar mats, the process of the invention could bemodified to use a different system to contact the tar mat with ozone byinjecting ozone into the water layer below the oil reservoir with itsbottom tar mat. The ozone is conveyed up through the water to the matlayer of solid polycyclic aromatic hydrocarbons (PAH) underlying thereservoir where it breaks the aromatic rings. The mat can prevent thehydrostatic pressure of the water from acting on the reservoir.Treatment causes the mat to become more permeable to water, allowing theunderlying hydrostatic pressure to act on the oil reservoir and aid inoil recovery. A solvent is preferred to stabilize the ozone until itreaches the mat layer. The solvent is immiscible in water, non-polar,lighter than water, and organic.

The ozone is injected in the water layer or aquifer that underlies thePAH tar mat at the bottom of an oil reservoir. Preferably, the ozone isinjected alone or in the form of a solution of a non-polar solvent. Theozone rises through the water aquifer (and possibly through thenon-polar solvent layer) and contracts the mat layer. There it attacksthe PAH to form polar products that are partitioned into the waterphase. Thus, the reaction of the ozone with the PAH compounds in the matlayer solubilizes these compounds and increases the permeability of themat layer. The increased permeability allows the hydrostatic pressure ofthe water to act on the oil reservoir above the mat layer, thusincreasing productivity of the oil reservoir.

The solvent helps convey the ozone to the mat layer and stabilized theozone. The solvent also solubilizes non-polar compounds in the mat,making them more available for reaction with the ozone. The solvent isbuoyant in water, and immiscible with water so that it will rise throughthe water to PAH layer. Suitable solvents are any buoyant material thatsolubilized PAH and non-polar materials, and are immiscible in water.Suitable solvents include, but are not limited to fully saturatedhydrocarbons, such as straight-chain or branch-chain hydrocarbons of 7or more carbons, and halogenated hydrocarbons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and 1 b. A schematic of a reactor setup used in the examples,(a) packed column reactor fed by an ozonated water reservoir; (b): batchreactor.

FIG. 2. Gas chromatographs of intermediates and products from pyrene ineffluents of: (a) column reactor prior to ozonation, (b) column reactorafter 1 hr of ozonation, and (c) batch reactor after 1 min of ozonation.Identified: compounds include 1-pyrene, 2-4,5-phenanthrenedialdehyde,3-2,2′,6,6′-biphenyltetraaldehyde, 4-1,2-benzenedicarboxylic acid,diisooctyl, 5-benzylbutyl phthalate, 6-diethyl phthalate,8-4H-cyclopenta[def]phenanthrene, 10-xanthone, 11-butylatehydroxytoluene, 2-dibutyl phthalate, 13-nonyl phenol, 16-hexadecanoicacid, 17-tetradecane, 19-hexadecane, 20-henicosane, 21-6-propyltridecane, 22-docosane, 23-hexacosane, 24-pentacosane, 26-unknown(m/z=154), and 27-unknown (m/z=139).

FIG. 3. Mass spectra of (a) 4,5-phenanthrenedialdehyde, and (b)2,2′,6,6′-biphenyltetraaldehyde.

FIG. 4. Schematic diagram illustrating concentration profiles ofreactants and intermediates in a flow-through column reactor.

FIG. 5. Products from pyrene in ozonated column effluents collected atdifferent time periods: (a) First 0.5 hr; (b) 0.5-1.0 hr; (c) 1.0-1.5hr; and (d) 1.5-2.0 hr. Identified: 1-pyrene,2-4,5-phenanthrenedialdehyde, 3-2,2′,6,6′-biphenyltetraaldehyde,4-1,2-benzenedicarboxylic acid, diisooctyl, 5-benzylbutyl phthalate,10-xanthone, 12-dibutyl phthalate, 20-henicosane, 21-6-propyl tridecane,22-docosane, 23-hexacosane, 26-unknown (m/z=154), and 27-unknown(m/z=139).

FIG. 6. Gas chromatograms of different intermediates from pyrene(suggestive of free-radical reactions) identified at different treatmentstages: (a) compounds 1-4 during first 15 min; (b) 5-10 in addition to1-4 during 15-30 min; and (c) 1-4 during 30-45 min. Identified:1-pyrene, 2-2,2′,6,6′-biphenyltetraaldehyde, 3-4,5-phenanthrenedialdehyde, 4-1,2-benzenedicarboxylic acid, diisooctyl, 5-henicosane(C₂₁), 6-docosane (C₂₂), 7-tricosane (C₂₃), 8-tetracosane (C₂₄),9-pentacosane (C₂₅), 10-hexacosane (C₂₆).

FIG. 7 a-7 d. Proposed degradation mechanism of pyrene illustratingproposed intermediates as well as identified intermediates and productsduring ozonation.

FIG. 8. Changes in BOD and COD throughout the 20-day biotreatment test(means of BOD triplicates and of COD duplicates with standard deviationbars).

FIG. 9. Measured toxicity of the ozonated column effluent throughout the20-day biotreatment test (means of duplicates with standard deviationbars).

FIG. 10. Intermediates and products identified at different stages ofthe 20-day biotreatment test (on the ozonated column effluent): (a)prior to biotreatment; (b) after 5-day biotreatment; (c) after 10-daybiotreatment; (d) after 15-day biotreatment; and (e) after 20-daybiotreatment. Identified: 1-pyrene, 2-4,5-phenanthrenedialdehyde,3-2,2′,6,6′-biphenyltetraaldehyde, 5-benzylbutyl phthalate, 10-xanthone,12-dibutyl phthalate, 16-hexadecanoic acid, 22-docosane, 26-unknown(m/z=154), 27-unknown (m/z=139), 28-phosphoric acid tributyl ester, and30-biological culture (m/z=226).

FIG. 11. Measured COD of the ozonated batch effluent throughout 50 min.of ozonation time, mean±standard deviation of triplicates shown.

FIG. 12. Intermediates and products identified and quantified in a batchreactor containing a benzo[a]pyrene suspension after being ozonated for(a) 2 min; (b) 10 min; (c) 30 min; and (d) 50 min. (All species arelisted in Table B-II.)

FIGS. 13 a to 13 d. Degradation pathways of ozonated benzo[a]pyrene: (a)overall pathways showing major identified intermediates and products;(b) proposed mechanistic steps in the formation of oxygenatedintermediates; and (c) proposed mechanistic steps in the formation ofaliphatic products.

FIG. 14. Biodegradation intermediates and products identified andquantified during a 20-day biological incubation of a pretreatedeffluent obtained from a benzo[a]pyrene-packed column fed withozone-laden water. (a) Before incubation; (b) after 5-day incubation;(c) after 10-day incubation; (d) after 15-day incubation; and (e) after20-day incubation. (All species are listed in Table B-II.)

FIG. 15. (a) BOD and COD changes during the 20-day biological incubationof the ozone-pretreated column effluent (as in FIG. 5); shown are meanand standard deviation of triplicate COD, and mean and range ofduplicate BOD. (b) Toxicity assessed during the 20-day biologicalincubation of the ozone-pretreated column effluent (as in FIG. 5), withmean and range of duplicates shown.

FIG. 16. A graph showing the ozonation results of various alkane andsaturated ring compounds.

FIGS. 17 a and 17 b. A graph showing the ozonation results of variousaromatic compounds

FIG. 18 A graph showing the ozonation results of alkyl benzenes in abipolar solvent.

FIG. 19 A graph showing the ozonation results of aromatic sulfurcompounds.

FIG. 20. A graph showing the results of ozonation of high-molecularweight and low molecular weight sulfur compounds.

FIG. 21. A graph showing the ozonation results of saturated andunsaturated fractions in waste oil.

FIGS. 22 a and 22 b. A graph showing the toxicity and BOD of variousfractions according to ozonation duration.

FIG. 23 A graph showing the solubility of ozone in various solvents.

FIG. 24 A schematic of an embodiment of the invention.

FIG. 25 Another schematic of another embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION Example A Degradation of pyrene

This example focuses on an integrated approach for the degradation ofpyrene involving chemical oxidation followed by biological treatment.The objectives were to: 1) provide mechanistic details in thedegradation of pyrene subject to ozone treatment, 2) test the combinedtechnique of ozone pretreatment followed by biological degradation, and3) test a pretreatment column to promote efficient use of chemicaloxidants and biodegradability. Batch and packed column reactors wereused to examine the degradation pathways of pyrene subject to ozonationin the aqueous phase. After different ozonation times, samplescontaining reaction intermediates and byproducts from both reactors werecollected, identified for organic contents, and further biologicallyinoculated to determine biodegradability. The O₃-pretreated samples wereincubated for 5, 10, 15, and 20 days, after which biochemical oxygendemand (BOD), chemical oxygen demand (COD), and toxicity tests alongwith qualitative and quantitative GC/FID and GC/MS analyses of pyrene,intermediates, and products were performed. Intermediates identified atdifferent stages included 4,5-phenanthrenedialdehyde,2,2′,6,6′-biphenyltetraaldehyde, and long-chain aliphatic hydrocarbons,which suggested that the degradation of pyrene was initiated by O₃ viaring cleavage at the 4,5- and 9,10-bonds and that further oxidationensued via reactions with both O₃ and OH. until complete mineralization.Intermediates formed during chemical oxidation were biodegradable with ameasured first-order rate constant (k₀) of 0.243 day⁻. The integratedchemical-biological system appeared to be feasible for treatingrecalcitrant compounds, and a chemical pretreatment column wasparticularly useful in promoting soluble intermediates from otherwisehighly insoluble, inaccessible pyrene.

Materials and Methods

Chemicals

Ozone (˜1% w/w ozone in air) was generated from filtered, dry air by anozonator (Model T-816, Polymetrics Corp.). Pyrene (99%, Aldrich ChemicalCo.) was washed with distilled-deionized (DD) water three times,extracted by dichloromethane (DCM), and the solvent evaporated by agentle stream of nitrogen gas. Stock and working indigo blue solutionswere prepared from potassium indigo trisulfonate (C₁₆H₇N₂O₁₁S₃K₃,Aldrich Co.) per Standard Methods (APHA et al., 1992a). Polyseed (HachCo.) was used in dilution water for biochemical oxygen demand (BOD)measurements per Standard Methods (APHA et al., 1992b). Inoculum fortoxicity test was prepared according to a Hach method (HACH,1988-1995b). COD digestion solutions (0-15,000 mg/L, 0-40 mg/L range,Hach Co.), ToxTrak™ reagent powder pillows, and ToxTrak™ acceleratorsolution (Hach Co.) were purchased and used according to themanufacturer's methods without further processing. Low-organic (<15 ppbas TOC), low-ion (resistivity>18 MΩ-cm), and non-pyrogenic (up to 4-logreduction with reverse osmosis pretreatment) DD water was used in allprocedures (4-stage Mill-Q Plus system, Millipore Co.). Dichloromethane(Fisher Scientific) of HPLC grade was used in liquid-liquid extractionprocedures. Other chemicals used in this research were of reagent grade.

Analytical Methods and Equipment

Aqueous concentration of ozone in the reactor was determined by sampleabsorbance at 600 nm using a 1-cm quartz cell with a IIP-8452Spectrophotometer (HP-8452 UV-Vis Spectrophotometer, Hewlett PackardCo.) according to the Indigo Blue Method (APHA, et al., 1992a). Thefollowing formula was used for our modified procedure based on weighing:

O₃[mg/L]=((SW+IW)/SW)×((DF×A _(blank))−As)/f

Where:

SW [g]=sample weight (W_(I+S)−W_(I)),W_(I+S) [g]=weight after adding indigo blue solution (7 mL) plus sample(˜3 mL),W_(I) [g] weight after adding indigo blue solution (7 mL),IW [g] weight of indigo blue solution (W_(I)−W_(emp)),W_(emp) [g]=weight of the empty test tube,A_(blank) [#]=absorbance at 600 nm of the indigo blue analyticalsolution without the sample,A_(s) [#]=absorbance at 600 nm of the indigo blue analytical solutionplus the sample,DF [#]=dilution factor, DF=IW/(SW+IW),f=0.42.

Sample COD determinations were made per Hach COD method (HACH,1988-1995a) using a COD reactor (Hach Co.) and a direct readingspectrometer (DR/2000, Hach Co.) or the HP-8452 spectrophotometer forultra-low range COD measurement at λ=356 nm. Sample BOD determinationswith required controls were made per Standard Methods (APHA et al.,1992b) using an oxygen meter/electrode system (YSI Model 57 oxygen meterwith oxygen electrode, YSI Co.). Sample toxicity was quantified based ona colormetric method of measuring the reduction of the redox-active dyeresazurin by bacterial respiration (HACH, 1988-1995b) with thespectrometer (DR/2000, Hach Co.).

Quantification of organics

Extraction. Typically a 200-ml sample containing pyrene and/or organicproducts was extracted three times using a total of 100 ml DCM. Thecombined extract was concentrated to 4 ml by evaporation using aKuderna-Danish evaporator (ACE glass Inc.) followed by furtherevaporation to 0.2 ml using a gentle stream of N₂ gas. The extract wasstored at −12° C. until analysis.

Quantification and identification. Extracted samples containing pyrene,intermediates, and products were analyzed using a gas chromatograph (GC)(HP 5890, Hewlett Packard Co.) equipped with a capillary column (RTX-1non-polar column, 30 m×0.25 mm×0.25 μm, Baxter Co.) and a flameionization detector (FID). The GC was interfaced and programmed with theHP Chemstation software (Hewlett Packard Co.). Quantification was basedon an external standard and calculation using a pyrene calibrationcurve. A 5:1 split injection was used with an oven temperature from 50°C. (1 min) to 300° C. (60 min) at a 5° C./min ramp.

Tentative identification of intermediates and oxidation products wereperformed using a GC (HP 6890) with a capillary column (DB-1 non-polarcolumn, 60 m×0.25 mm×0.25 μm, J & W Co.) and a mass spectrometrydetector (MS) (HP 6890) interfaced and programmed with the HPChemstation software (Hewlett Packard Co.). A split ratio of 5:1,solvent delay at 12 min, and scan range from m/z 15 to m/z 500 at 1.4scan/sec were used. The oven temperature was set from 50° C. (1 min) to300° C. (60 min) at 5° C./min ramp. The HP Chemstation library (HewlettPackard Co.) was used for species identification as a supplement to massspectral and retention time characteristics. All library-matched speciesexhibited the degree of match better than 90%. In addition, comparisonof parent compound structure and interpretation of mass spectra of theintermediates from ion fragmentation information were performedparticularly for the identification of key intermediates4,5-phenanthrenedialdehyde and 2,2′,6,6′-bephenyltetraaldehyde.

Reactors and Procedures

Reservoir of Ozonated Water

Ozone, generated at an applied voltage of 120 V and air flow rate of 2L/min, was spared into a mixed flow-through reservoir (CSTR type)holding a water that was slowly overflowing but at a constant volume ofabout 3 L (FIG. 1). The pH of this reservoir was maintained at 7 byautomatic delivery of concentrated NaOH via a peristaltic pump connectedto a pH probe/meter/controller system (Cole Parmer Co.). Duringozonation, slowly overflowing (50 mL/min) water was passed through,ozonated in, the reservoir and the dissolved ozone concentration wascontinually monitored. After the aqueous ozone reached the steady-stateconcentration, the ozonated water was introduced into the packed columnreactor by a peristaltic pump (Masterflex computerized drive, ColeParmer Co.) (FIG. 1).

Packed Column Reactor

Weighted glass beads (˜150 g) of ca. 1-mm diameter were washed withconcentrated K₂Cr₂O₇/H₂SO₄, concentrated HNO₃, DD water, acetone, andDCM sequentially, then dried at 400° C. overnight. About 1 g of pyrenewas weighted and dissolved into 20 ml DCM, the solution was added withthe pretreated glass beads. The mixture was agitated and DCM evaporatedcompletely by blowing of N₂ gas. The glass beads mixed withrecrystallized pyrene solid were packed into a glass column(Adajusta-Chrom 0.9839″×300 mm glass column, ACE glass, Inc.). Thelength of the packed zone was about 7.5 in. During the course ofreaction, water carrying dissolved O₃ was passed through the packedcolumn in the upflow direction using a peristaltic pump at 44 mL/min, asshown in FIG. 1. Samples were collected at the column outlet at varioustime intervals, filtered through a 0.45-μm filter, and analyzed for O₃as well as organic contents. Tests of BOD₅, 20-day BOD, COD, toxicity,and qualitative and quantitative analyses of pyrene, intermediates, andproducts of both chemical and biological treatments were performedsimultaneously.

Batch Reactor

A glass batch reactor with a working volume of 1,700 mL was used (ACEglass Inc.). Mixing of this reactor was provided by two TEF agitators(ACE glass Inc.) driven by a variable speed controller/motor (ACE glassInc.) through a flexible drive cable. Ozone gas was sparged into thereactor near the bottom through a glass dispersion tube (ACE glass Inc.)Constant pH during reaction was maintained at 7 automatically. Afterabout 1 g of pyrene solid was added into the reactor filled with1,700-ml water, ozone was sparged into the batch. The dissolved ozoneconcentration was continually monitored. Samples were collected after 2,4, 6, 8, 10 min of ozonation and filtered through a 0.45-μm filter.Tests of BOD₅, COD, toxicity, and qualitative and quantitative analysesof pyrene, intermediates, and products before and after chemical andbiological treatments were performed simultaneously.

Results and Discussion

Ozonation of pyrene was carried out in batch and column reactors tostudy: 1) the effect of reactor on intermediates and products formation,2) the degradation pathway of pyrene under ozonation, 3) thebiodegradability of intermediates, and 4) the feasibility of a combinedchemical-biological treatment system for pyrene. Reaction solutionsduring ozonation and biodegradation processes at different stages werecollected and the intermediates and byproducts identified by GC/MStechniques.

1. Effects of the Reactor Type on Intermediates and Products Formation

To delineate the influence of reactor configurations on the formation ofintermediates and products, ozonation experiments using aqueous andexcess pyrene were carried out in batch and packed column reactors. BOD₅and COD were measured for three ozonated, filtered solutions: 1) asaturated aqueous solution of pyrene (0.13 ppm), 2) the solution afterozonation of an excess pyrene suspension (1 g/1.7 L), and 3) theeffluent of a column packed with excess pyrene solid (1 g) and glassbeads (7.5 in. in bed-length). The saturated pyrene solution wasprepared by allowing excess pyrene solid to reach dissolutionequilibrium in water overnight followed by removal of the excess solidusing a 0.45-μm filter. The ozonated batch solution was obtained after10 min of ozonation and filtered, while the effluent was collected fromthe packed column fed with ozonated water over a 4-hr period. Table A-Ishows the results of BOD₅ and COD measurements. The BOD₅ for thesaturated pyrene solution approximates over 80% of the COD value,suggesting that pyrene in its dissolved form is amenable tobiodegradation, albeit in small quantity. The aqueous phase COD from theozonated batch reactor increased after ozonation possibly due tooccurrence of intermediates or pyrene-derivatives that are more solublein water as a result of ozonation. The new, lower BOD₅/COD ratio of 66%appeared to suggest either that a larger amount of degradable substrateswas available after ozonation that resulted in lower BOD₅, or morelikely that the biodegradability of the ozonated solution decreased as aresult of ozonation possibly due to formation of slightly morerecalcitrant intermediates. Following the reasoning of increased aqueousCOD due to abundance of more soluble intermediates, the measured COD forcolumn effluent would imply that it contained much more intermediatesand byproducts. The new BOD₅/COD ratio registered a slightly smallervalue of 0.53. These ratios are well within those commonly observed fordomestic wastewater and do not seem to signify toxicity.

Parallel to BOD₅ and COD measurements of the ozonated reaction media,the effects of reactors on intermediates formation were further probedusing GC/FID and GC/MS identification techniques. FIG. 2 shows the gaschromatograms of parent and identified intermediate compounds in 1) theaqueous pyrene solution without ozonation, 2) ozonated column effluent,and 3) ozonated batch solution. Despite its low solubility, the parentpyrene (peak 1 as labeled) was found in all solutions. As listed inTable A-II, twenty-five other compounds were found in the columneffluent, and except for two of them were identifiable by MS librarycomparison. Two important intermediates 4,5-phenanthrenedialdehyde(species 2) and 2,2′,6,6′-biphenyltetraaldehyde (species 2) were foundin the ozonated column effluent but not in the ozonated batch solution.The mass spectra of these two intermediates, species 2 (m/z 234) and 3(m/z 266), are shown in FIGS. 3( a) and (b), respectively.

In the ozonated batch solution, found in place of the di- andtetra-aldehydes was a variety of benzenediacarboxylic acids, whichapparently are subsequent byproducts in the oxidative chain of events.Comparison of gas chromatograms (b) and (c) of FIG. 2 shows that thecolumn effluent contained an abundance of intermediates (such as species2 and 3) whereas the ozonated batch solution contained lessintermediates but more fragments that were products further down thedegradation process. These identifications are consistent with thehigher COD measurement in the column effluent than that in the batchsolution.

These results indicated that ozone was capable of degrading pyrene viaring opening, as evidenced by intermediates dialdehyde and tetraaldehyde(species 2 and 3 in the column effluent), and further oxidation by ozone(and other oxygenated radicals to be discussed) to other fragments andbyproducts (such as 1,2-benzenedicarboxylic acid, diisooctyl 4,benzylbutyl phthalate 5, hexacosane 23, henicosane 20, and nonyl phenol13 in the batch solution) if the intermediates were to remain exposed toozone. These results underscored the importance of the role that thereactor configuration played in determining the kinds and amounts ofintermediates and byproducts to be found after ozonation. The influenceof a column reactor on the types and amounts of intermediates andbyproducts formed are illustrated in FIG. 4. A batch reactor readilysubjects the intermediates from pyrene to continual O₃ attack andfurther degradation, whereas the column reactor allows the intermediatesto be eluted from the O₃-rich area, i.e., the reactive zone. Thus, topromote the formation of intermediates that could be subsequentlybiodegraded rather than relying upon ozone as the sole oxidant in thecomplete degradation of pyrene, a column reactor was used to collecteffluent that was rich in partially treated intermediates for furthermechanistic and biodegradability studies.

Degradation Pathway of Pyrene in Ozonated Water

The effluent from a pyrene-packed column fed with ozonated water wascollected and identified for intermediates and byproducts via GC/FID andGC/MS. The up-flow influent water contained 5 mg/L O₃ while the effluentnone, indicating that complete consumption of O₃ occurred in the column.The filtered (through 0.45 μm) effluents exhibited yellowishintermediate compounds that were not apparent in previous samples fromozonated batch solutions. The absence of colored compounds in the batchreaction was attributed, as explained previously, to continualdegradation of the colored intermediates by O₃. FIG. 5 identifiedspecies found in the effluents collected at different time intervals.These identified species, including the dialdehyde (2) and tetraaldehyde(3) intermediates, resembled those identified in FIG. 2. In addition tothe molecular ion peaks, other fragments including m/z 205, 176 and 29corresponding to the loss of —CHO groups were noticeable in the massspectra in the case of 4,5-phenanthrenedialdehyde, and m/z 237, 29 of2,2′,6,6′-biphenyltetraaldehyde. A biphenyl fragment was found at m/z152 in FIG. 3( b), which suggested the presence of a biphenyl structureas 2,2′,6,6′-biphenyltetraaldehyde.

FIG. 2 a showed a substantial variety of (unozonated) compounds elutedfrom the column even prior to the start of ozonation. These compoundswere in many cases similar to that after ozonation as shown in FIG. 5 a.This was attributed to the occurrence of autooxidation (reaction withmolecular oxygen), which is also an oxidation process albeit at a muchslower rate than of oxidation by ozone, resulting in similarintermediates and products. Autooxidation of pyrene could have occurredduring storage on the shelf or by dissolved oxygen after being dispersedand thinly packed in the column. The latter was more likely ascalibration runs prior to column loading did not reveal theintermediates. However, clearly discernable was that these intermediateand product peaks intensified pronouncedly after the ozone oxidant wasintroduced, as evident in comparison of FIG. 5 a with FIG. 2 a. Dataestimation suggested that oxygenated compounds such as 5, 8, 9, 11, 12,13, and 16 increased by 1290%, 1160%, 690%, 1130%, 60%, 20%, and 200%,respectively, while aliphatic compounds such as 17, 18, 20, 22, 23, and24 increased by 410%, 1410%, 3530%, 3530%, 2670%, 4140%, respectively.

FIG. 6 identified intermediates found in ozonated column effluentscollected by more frequent samplings. The gas chromatograms as shownfocused on species with retention times of 40-50 min, which consistedmainly of pyrene, 2,2′,6,6′-biphenyltetraaldehyde (2),4,5-phenanthrenedialdehyde (3), and 1,2-benzenedicarboxylic acid,diisooctyl (4). In addition to these major species, spectrum (b) of FIG.6 also identified long-chain aliphatic carbon compounds, including C₂₁,C₂₂, C₂₃, C₂₄, C₂₅, and C₂₆, shown as species 5 to 10, respectively.These long-chain hydrocarbons disappeared as the column effluentestablished steady-state levels of intermediates; henceforth only fourmajor intermediates (1 to 4) remained after 45 min., as shown bychromatogram of FIG. 6( c). The presence of long-chain aliphatichydrocarbons that have more carbons than the 16-C pyrene parent issuggestive of, during ozonation, the involvement of free-radicalpathways in which radical recombinations are prevalent. The decomposingof O₃ in water is known to occur through a series of free radical chainreactions that involve reactive radicals including OH./O.⁻, HO₃./O₃.⁻,and HO₂./O₂.. These reactive radicals are potent oxidants that can reactwith organic molecules leading to their mineralization.

With identified intermediates and byproducts, in FIGS. 7 a and 7 b (thetop of 7b continues from the bottom of 7a) is shown a proposed mechanismdepicting the degradation pathway of pyrene under ozonation. As shown,the degradation was initiated by electrophilic attack of O₃ on one ofthe electron-rich conjugate rings of the pyrene molecule resulting inthe formation of dialdehyde (2) and, upon another ring-opening attack,tetraaldehyde (3). Pyrene has an asymmetrical fused-ring structure. Thebonds between fused angular rings, as in 4,5- and 9,10-bonds (referredto as the K-regions), have the highest bond order (0.833) and shortestbond length (1.367) in the pyrene molecule (Harvey, 1997). These bondsshow considerable double-bond character and are more reactive than otherbonds, consistent with K-regions being the first activated reactionsites in metabolic oxidation. The preferential attack of O₃ on the4,5-bond of the pyrene molecule is also explained in terms oflocalization energy that marks the site as being most reactive (Bailey,1982). Subsequent reactions of intermediates with O₃ or oxygenatedradicals (e.g., OH., O₂.⁻, O₃.⁻) resulted in additional intermediates(4)-(16). The production of long-chain aliphatic hydrocarbons, compounds(13) through (25), was attributed to oxidation reactions prompted by O₃,OH., and other free radicals. With proposed intermediates and identifiedones, FIGS. 7 c and 7 d details the formation and destruction of some ofthese compounds based on known reaction pathways reported in theliterature. As shown, these reactions produced alkyl radicals thatfurther propagated chain reactions and eventually led to polymerizationvia recombination of the organic radicals. Thus, the formation of manyoxygenated intermediates (4, 7, 9, 12, 14, 16) as well as n-alkanes (19,22) could be accounted for by FIGS. 7 c and 7 d.

Another observation supporting the involvement of free radicals was thedisappearance of these long-chain alkanes if the effluent was subject toprolonged ozone hydrolysis. Long-chain alkanes are characteristicallyresistant to electrophilic attack by O₃ yet susceptive to OH. oxidation.Simpler short-chain polar aliphatic compounds were expected but notfound in the reaction mixture; their absence was attributed toanalytical extraction and preparation procedures that failed to retaincompounds with less than six carbons. As O₃ undergoes hydrolysis duringozonation, both O₃ and OH. are available for the degradation of pyrene.It is plausible that the degradation pathway is initiated mainly viaring opening by O₃, continued in fragmentation by both O₃ and OH., andultimately brought to complete mineralization primarily via OH.radicals.

Biodegradation of Ozonated Column Effluent

The biodegradability of the ozonated column effluent was tested byincubating the effluent over a 20-day period throughout which the CODand BOD of the flasks were monitored. FIG. 8 shows the measurementstaken after 0, 5, 10, 15, and 20 days. The results indicated an increaseof BOD from 0 to 4.2 mg/L during the first 10 days and leveling off overthe remaining. The measured COD exhibited a complimentary curve showinga decrease in COD from 7.0 mg/L to 3.1 mg/L over the first 10 days and aconstant level afterward. These results suggested that biodegradableorganic compounds in the effluent were biodegraded over the first 10days. The BOD curve was fitted with first-order kinetics using theleast-square method with a first-order rate constant k₀=0.243 day⁻¹ andan ultimate BOD L₀=4.25 mg/L. The obtained value of k₀ approximatesclosely that of domestic wastewater routinely treated by biological unitprocesses.

The acute aqueous toxicity during the 20-day incubation period was alsomonitored using a standard effluent toxicity test (HACH, 1988-1995b).FIG. 9 shows the percentage inhibition value (% inhibition) of theincubated samples over the same intervals. The measurements registeredinhibition values within ±10% that was within the nontoxic range of themethod. This means that the effluent was nontoxic to the receivingE-Coli bacteria, and that the effluent contained biodegradableintermediates and byproducts, including biodegradation products, whichpossessed no acute toxic effects to the bacteria.

Concurrent to measurements of COD, BOD, and toxicity was the GC/FID/MSidentification of various intermediates and byproducts present in theflask over the same incubation period. FIG. 10 shows compounds foundafter 0, 5, 10, 15, and 20 days of incubation. The changes of speciationin the incubated effluent are more clearly tracked by Table A-III. Thedisappearance of 11 intermediates, including species 1, 3, 7, 8, 10, 13,15, 16, 19, 21, and 22, during the first 5 days of incubation was mostnotable. Dissolved parent compound pyrene (1) and intermediatesdialdehyde (2), tetraaldehyde (3), and other benzenedicarboxylic acidseither disappeared or decreased in concentrations over the incubationperiod. The significant disappearance of many intermediates in the first5 days is consistent with the much more rapid changes in BOD and CODduring the initial period. After 5 days, virtually all other compoundsremained detectable throughout the incubation, which signaled that thesecompounds could not be further biodegraded, consistent with relativelymild changes in BOD and COD. Also detected after 15 and 20 days was thephosphoric acid tributyl ester with a structure similar to high-energyphosphoanhydride bonds, which indicated that biosynthesis of ATP mighthave occurred along with the biodegradation processes.

Oxidant Balance for Column Ozonation

To determine the efficacy of ozone treatment, COD contents in apyrene-packed column and in the effluent before and after ozonation weremeasured to establish a COD balance. In this experiment, the column waspacked with glass beads and 0.147 g pyrene that amounted to a totaldemand of 432 mg O₂ as determined by COD test. Ozonated water was elutedthrough the column at 44 mL/min over 4 hours with a total throughput of10.5 L. Influent and effluent O₃ concentrations were frequently measuredat 5.05 mg/L and 0.0 mg/L, respectively; major parent and intermediatecompounds, i.e., pyrene, 4,5-phenanthrenedialdehyde, and2,2′,6,6′-biphenyltetraaldehyde, in the effluent and in the columnbefore and after ozonation were quantified. The results are shown inTable A-IV.

The total amount of O₃ consumed in this 4-hr experiment was 1.1 mmol or53.0 mg (i.e., 5.05 mg/L×10.5 L), which would mineralize up to 0.09 mmolor 18.1 mg pyrene according to this stoichiometric equation:C₁₆H₁₀+12.3O₃=16CO₂+5H₂O. The amount of O₃ consumed could reduce the CODof the system by 18 to 53 mg of O₂ demand depending on the number ofoxygen atoms of O₃ involved in the oxidation. The total amount of pyrenedegraded in this experiment was 73.8 mg or 0.365 mmol. The mole ratio ofconsumed ozone to consumed pyrene was 1.1/0.365=3.3; thus, three molesof ozone were consumed for each mole of pyrene degraded. This observedratio of 3.3 is clearly lower than that of 12.3 theoretically requiredfor the complete mineralization of pyrene. Thus, significant amounts ofintermediates and byproducts would be expected either in the effluent oras residuals in the column, which were indeed observed and evidenced bythe higher measured COD due to intermediates in the effluent.

The COD measurements of Table A-IV also indicated a reduction of COD inthe system (column residual plus effluent) by 36 mg O₂. This value lieswell within 18 to 53 mg O₂ afforded by O₃ over the experiment duration.This means that the supplied O₃ was primarily consumed in convertingparent pyrene to intermediates thereby reducing system COD, and that O₃had not been wasted in decomposing via hydrolysis.

From the viewpoint of applying biological treatment following ozonation,it is desirable to have a lower ratio in consumed ozone to consumedpyrene but higher COD and BOD values in the effluent. Such a system willchemically pre-treat the largely insoluble pyrene to dissolvedintermediates that are accessible and effectively biodegraded. Itappears that an ozonated column pretreatment system can be better tunedto produce more intermediates for a sequential chemical-biologicaltreatment system than a batch reactor that rely more on O₃ to mineralizeintermediates.

Conclusions

This example examined the feasibility of an integratedchemical-biological system for the treatment of highly recalcitrantpyrene. The refractory nature of pyrene was thought at least in part dueto its low solubility that limited access by microbes. Despite limitedwater solubility, pyrene can be made more soluble if one or more of itsfused rings were hydroxylated with hydroxyl group (—OH) or cleaved withaldehyde group (—CHO), i.e., pyrene can be transformed into more solublederivatives by reaction with OH. resulting in hydroxylation or with O₃resulting in ring cleavage. Whereas in a batch reactor O₃ and itsradical oxidants are capable of mineralizing pyrene and its derivatives,a column ozonation system makes more effective use of O₃ by generatingmore oxidation intermediates that can be subsequently biodegraded. Thisis evident from that the column effluent contained 4 times as much CODand 90% of which was biodegraded with 10 days. Mechanistically, thedegradation of pyrene under ozonation was found, as supported byidentified intermediates and byproducts, to proceed via initial ringcleavage by O₃ at the 4,5- and 9,10-bonds and continued oxidation by O₃and OH.. For otherwise scantly accessible pyrene, the combinedchemical-biological treatment scheme appears to promote efficient use ofchemical oxidant in pretreatment and effective biodegradation of thenontoxic, abundant, biodegradable intermediates.

Example B Degradation of benzo[a]pyrene

This example focuses on an integrated treatment of benzo[a]pyreneinvolving sequential chemical oxidation and biological degradation. Theobjectives are to: 1) provide mechanistic details in the ozone-mediateddegradation of benzo[a]pyrene in the aqueous phase, 2) test thebiodegradability of resultant intermediates, and 3) test the feasibilityfor the coupled chemical-biological treatment of the 5-ring PAH. Batchand packed column reactors were used to examine the degradation pathwaysof benzo[a]pyrene subject to ozonation in the aqueous phase. Afterdifferent ozonation times, samples containing reaction intermediates andbyproducts from both reactors were collected, identified for organiccontents, and further biologically inoculated to determine theirbiodegradability. The O₃-pretreated samples were incubated for 5, 10,15, and 20 days; afterward biochemical oxygen demand (BOD), chemicaloxygen demand (COD), and E-Coli toxicity tests were conducted along withqualitative and quantitative determinations of benzo[a]pyrene,intermediates, and reaction products by GC/FID and GC/MS methods.Prevalent intermediates identified at different stages includedring-opened aldehydes, phthalic derivatives, and aliphatics. Thedegradation of benzo[a]pyrene is primarily initiated via O₃-mediatedring-opening, followed by O₃ and hydroxyl radical fragmentation, andultimately brought to complete mineralization primarily via hydroxylradicals. Intermediates formed during chemical oxidation werebiodegradable with a measured first-order rate constant (k₀) of 0.18day⁻¹. The integrated chemical-biological system seems feasible fortreating recalcitrant compounds, while pretreatment by chemicaloxidation appears useful in promoting soluble intermediates fromotherwise highly insoluble, biologically inaccessible benzo[a]pyrene.

Materials and Methods

Descriptions of sections on Chemicals, Analytical Methods and Equipment,and Reactors and Procedures were identical to Example A. Only deviationsfrom Example A are highlighted here. Benzo[a]pyrene (BaP) (98%, AldrichChemical Co.) in place of pyrene was used and purified as described. Atypical sample size for analysis is 150 ml and the storage temperatureawaiting analysis −12° C. With the same GC/MS system, a split ratio of5:1, solvent delay at 6 min, and scan range from m/z 15 to m/z 500 at1.4 scan/s were used. Comparison of parent compound structure andinterpretation of mass spectra of the intermediates from ionfragmentation information were performed particularly for theidentification of key intermediates 7-propanal-8-methylpyrene,7-ethyl-8-ethanalpyrene, and 4-methyl-5-hydroxylchrysene. Reactorsystems (FIG. 1) were identical to ones previously used except that 0.15g benzo[a]pyrene was prepared and loaded into the packed column reactor.Samples during batch reaction were taken at 2, 10, 20, 30, and 50 min.Sample BOD and toxicity were determined in triplicates and duplicates,respectively. Previous analytical efforts for pyrene were redirectedtoward benzo[a]pyrene.

Results and Discussion

The degradation pathway, biodegradability of intermediates, and oxidantbalance during ozonation of BaP will be addressed in turn.

Degradation Pathways of Ozonated Benzo[a]pyrene

COD measurements were made for three solutions: 1) a saturated aqueoussolution of BaP, 2) the solution after ozonation of a batch of excessBaP suspension (0.150 g/10.7 L), and 3) the effluent of a column packedwith excess BaP solid (0.149 g) and glass beads (7.5 in. in bed-length).The saturated BaP solution was prepared by allowing excess BaP solid toreach dissolution equilibrium in water overnight followed by removal ofthe excess solid using a 0.45-μm filter. The ozonated batch solution wasobtained after 50 min of ozonation and filtered, while the columneffluent was collected from the packed column fed with an ozonated waterover a 4-hr period and filtered. Table B-I shows the results CODmeasurements of all solutions and one BOD₅ measurement for the columneffluent. The saturated solution of BaP, due to its very limited aqueoussolubility, registered a negligible COD value compared to that of theozonated batch solution or the ozonated column effluent. In both thebatch and column solutions, much higher COD values were measured afterozonation, which indicated dissolution of daughter compounds of BaP intothe aqueous phase as a result of ozonation. A relativbiochemical oxygendemand ely high BOD₅-to-COD ratio of 0.43 was observed for the columneffluent, which suggested the intermediates were susceptible tobiodegradation, a point of further discussion later.

The COD values in the batch solution were relatively stable at about 15mg/L during the 50-min ozonation period, as shown in FIG. 11. Thisseemingly steady-state level of COD could be indicative of therelatively constant quantity of intermediates that were continuallyadded to the aqueous phase via oxidation of the parent BaP solid, aswell as continually being removed via further mineralization by ozone.

The aqueous intermediates after ozonation were identified and quantifiedby GC/MS techniques. Over sixty compounds were identified asintermediates and products in this example. Table B-II lists theidentified compounds in the order of increasing retention time in the GCcolumn, and labels them numerically in the like order. Among the myriadof those identified are five intermediates including ring-openedaldehyde (28), phthalic derivatives (29 and 38), and alkane/alkene (34,12). These products would likely abound at different stages ofozonation, i.e., with the aldehyde and acid more prevalent in theinitial stage of ozonation and the alkene and alkane the later stage.

FIG. 12 shows the identified, quantified species during 50-min ozonationof a BaP batch suspension. Salient of this figure is the largelyabsence, particularly beyond initial minutes, of compounds with longercolumn retention time (e.g., >20 min; or compound 27 or higher) that aretypical of intermediates found in the early stages of ozonation orshortly after ring-opening of BaP. The absence is indicative of furtheroxidation of early intermediates such as phthalic acids into otherproducts. Furthermore, that those compounds with shorter columnretention times (e.g., compound 26 and lower) remained relativelyconstant over the ozonation period was consistent with the relativelystable COD measurements of FIG. 11 shown for the same period. Long-chainaliphatic alkanes such as compounds 58 to 61 eventually disappeared withozonation treatment longer than 2 minutes, as they were likelyfragmented by secondary free-radical oxidants such as the OH..Therefore, FIG. 12 indicates a steady-state conversion of the excess BaPsolid into more water-soluble intermediates such as aldehydes and acidsthat are rapidly converted to various alkane and alkene mainly byradical reactions discussed below. That, the reaction rates of oxidants(both O₃ and secondary oxidant OH.) with the earlier intermediates suchas oxygenated compounds being relatively faster than those with laterintermediates such as alkenes and alkanes, would explain the absence ofthe former intermediates but an abundance of the latter during theseemingly steady-state mineralization.

FIGS. 13 a and 13 b outline (with the top of 13b starting at the bottomof 13a) a general degradation pathway of BaP subject to ozonation in theaqueous phase based on actual identified compounds. In general, theearlier reaction stage is populated with aromatic, oxygenatedintermediates, while the latter stage with alkenes and alkanes. In moredetails, FIGS. 13 b and 13 c proposed mechanistic steps leading to theformation of various oxygenated intermediates (Sequences I to IV) andaliphatic compounds (Sequences V and VI), respectively. The underlined,numerated species were identified whereas the curly-bracketed ones wereproposed intermediates. As shown in FIGS. 13 a and 13 b, the degradationwas initiated by electrophilic attack of O₃ on one of the electron-richconjugate rings of the BaP molecule resulting in the formationring-opening products 27 and aldehydes 28 and 30. Subsequent reactionsof intermediates with O₃ or its concomitant oxygenated radicals (e.g.,OH., O₂.⁻, O₃.⁻) resulted in additional oxygenated intermediates such as36, 35, 29, 22, 32, 33. The production of alkenes (e.g., compounds 2, 3,4, 5, 6, 7, 9, 11, 14, 16, 17, 19, and 21) and long-chain aliphaticalkanes (e.g., compounds 60, 61, 31, 58) was attributed to oxidationreactions prompted by O₃, OH., and other free radicals. It should benoted that the formation of secondary radical oxidants including O₂.⁻,O₃.⁻, OH. as well as H₂O₂ resulting from hydrolysis of O₃ has beenextensively documented.

FIGS. 13 b and c explain the formation of observed products (speciesnumbers underlined) via proposed intermediates (shown in curlybrackets), many of which have been reported as plausible elsewhere andtheir reaction steps are cited with italicized numerals. Sequence I ofFIG. 13 c shows upon ozonation of BaP the formation of7-methyl-8-prypanal-pyrene (28) via epoxidation at 7,8-bond, followed bybond breakage resulting the aldehyde, followed by further epoxidation atthe 9,10-position resulting in the dihydrodiol that further reacts withO₃ and with loss of H₂O₂ ultimately leads to compound 28. The 4,5- and7,8-bond cleavage products (30 and 28, respectively) were found in thisexample. These bonds have lowest localization energy and thus are sitesmost susceptible to epoxide formation.

Sequence II produces phthalic anhydride (29) via 1,6-quinione ofbenzeno[a]pyrene and 1,2-anthraquinonedicarboxylic acid intermediates.Further ozonation of intermediates results in the primary ozonidestructure and secondary peroxidic intermediates, phthaladehydic acid,then ultimately phthalic anhydride 29.

Sequence III suggests that continued ozonation of phthalic anhydride 29leads to identified intermediates 35 and 36 via phthalic acid and itsradical that subsequently recombines with other alkyl radicals. Thebreakage of fragile R—O bonds in 35 and 36 further leads to 32 and 33.Ozonation of phthalic anhydride 29 can also cleave the molecule at the1,2-position, resulting in a primary ozonide structure. Alternatively,Sequence IV suggested continued ozonation of phthalic anhydride leads toa primary ozonide and peroxidic intermediates (as in Sequence III),followed by loss of —CO₂ and —CO groups resulting in the formation of1,3-diene intermediate, which upon renewed O₃ attacks as shown leads tothe formation of ester 22.

Sequence V of FIG. 13 d shows ring-opening products as phthalic acidderivatives, which upon OH. attack form alkene radicals. These radicalsundergo additional free-radical reactions with other alkene fragments,and their eventual radical recombinations lead to alkene products suchas 7 and 19. Appearing prima facie puzzling was an abundance of alkenesand alkanes observed amid the panoply of oxidized products in theozonated, highly oxidizing environment, which might have suggested themproducts of reduction reactions. Similar products were observed inExamples A for the ozonation of pyrene. Other alkanes, alkenes, andrelated compounds were previously observed as products from ozonation ofhydrocarbons as well. Nonanal and nonanoic containing straight chaincarbons were reported as oxidation products from ozonation of PAHs (20,21). Decane, decene, and epicosane were obtained from ozonation of1-dodecene (38). These aliphatic compounds were attributed to freeradical mechanisms at work. It is well accepted that as O₃ undergoeshydrolysis during ozonation, both O₃ and OH. are available for reactionswith species in the reaction medium. Sequence VI proposes apolymerization pathway for the formation of long-chain alkanes by theactions of O₃ and OH. radical. As shown, it is initiated by ring openingof intermediates such as 29, followed by fragmentation into ethene anddiene that undergo ionic and/or radical polymerization in the presenceof OH., resulting in identified alkanes such as tridecane (34) andhenicosane (61).

An observation supporting the involvement of free radicals was thedisappearance of these long-chain alkanes if the effluent was subject toprolonged ozone hydrolysis. Long-chain alkanes are characteristicallyresistant to electrophilic attack by O₃ yet susceptive to degradation byOH. produced via O₃ hydrolysis. Simpler short-chain polar aliphaticcompounds were expected but not found in the reaction mixture; theirabsence was attributed to analytical extraction and preparationprocedures that failed to retain compounds with less than six carbons.

As already mentioned, the presence of organic co-solvent and theconcentration of O₃ play an important role in determining productformation and distribution.

In summary, this example demonstrates that the degradation mechanism ofBaP is, as reconstructed based on about 60 observed intermediates andproducts, initiated primarily via ring-opening by O₃ at the onset,continued in fragmentation by both O₃ and OH., and ultimately brought tocomplete mineralization primarily via OH. radicals.

Biodegradation of Intermediates from Ozonated Benzo[a]pyrene

The biodegradability of intermediates resulting from ozonation of BaPwas tested by incubating the ozonated column effluent for a 20-dayperiod throughout which the intermediates were qualitatively andquantitatively determined. The effluent was collected from a BaP-packedcolumn fed with ozonated water. The up-flow influent water contained4.77 mg/L O₃ while the effluent none, indicating that completeconsumption of O₃ occurred in the column. FIG. 14 identifiesintermediates and byproducts present in the flask throughout theincubation period at 0, 5, 10, 15, and 20 days. At day-0, the speciationin the column effluent already differs from that in the ozonated batchsolution. The column effluent contains a larger presence of compounds 30and higher (i.e., early-stage intermediates with longer GC-columnretention times), which are attributed to the flow-through columnconfiguration. A batch reactor readily subjects the intermediates frompyrene to continual O₃ attack and further degradation, whereas thecolumn reactor allows the intermediates to be eluted from the O₃-richarea, i.e., the reactive zone. Thus, the column effluent contained alarger abundance of intermediates and the contents of the partiallytreated intermediates were chosen for test of their biodegradability.

FIG. 14 appears to show that the early products of ozonation (e.g.,Compounds 25 or higher) either decreased over the incubation period ordisappeared, whereas the later products such as alkenes 5 and 7 appearedto increase from 0 to 10 days but decrease or disappear by day-20.Overall, most intermediates and by-products in the column effluenteither significantly decreased or disappeared by the end of the 20-dayincubation period. It should be noted that compound 24,decafluorobiphenyl, was the added internal standard for the GCanalytical procedures. Therefore, the results seem to suggest thatozonation makes available from otherwise highly insoluble, inaccessibleBaP a plethora of water-soluble intermediates that are biodegradable.

Concurrent to GC identification and quantification procedures during theincubation period, measurements of COD, BOD, and toxicity were made.FIG. 15 a shows the BOD and COD changes after 0, 5, 10, 15, and 20 days.The results indicated an increase of BOD from 0 to and leveling at 2mg/L over the 20-day period. The measured COD exhibited a complimentarycurve showing a decrease in COD from 5.5 mg/L to 2.2 mg/L over the sameperiod. These results suggested that biodegradable organic compounds inthe ozonated column effluent were biodegraded over the incubationperiod, consistent with the quantification results of GC of FIG. 14. TheBOD curve was fitted with first-order kinetics using the least-squaremethod with a first-order rate constant k₀=0.18 day⁻¹ and an ultimateBOD L₀=2.2 mg/L. The obtained value of k₀ approximates that of domesticwastewater routinely treated by biological unit processes.

The acute aqueous toxicity during the 20-day incubation period was alsomonitored using a standard effluent toxicity test described previously.FIG. 15 b shows the percentage inhibition value (% inhibition) of theincubated samples over the same period at 0, 3, 4, 5, 10, 15, and 20days. The heightened acute toxicity at day 5 (−19%) appeared to be anoutlier as it was not supported by speciation and quantification resultsof FIG. 14. In general, the measurements registered inhibition valuesmostly within ±10% that was within the nontoxic range of the method.This means that the effluent was nontoxic to the receiving E-Colibacteria, and that the effluent contained biodegradable intermediatesand byproducts, including biodegradation products, which possessed noacute toxic effects to the bacteria.

The integrated chemical-biological system being investigated is usefulfor treating highly recalcitrant BaP, the refractory nature of which hasbeen thought at least in part due to its low solubility that limitedaccess by microbes. The present process rendered BaP more soluble andthus biologically accessible by cleaving one or more of the fused ringsresulting in intermediates containing aldehyde (—CHO) or carboxylic(—COOH) groups, i.e., BaP was transformed into water-soluble,biodegradable derivatives by reaction with O₃ and secondary radicaloxidants.

Oxidant Balance for Column Ozonation

To determine the efficacy of ozone treatment, COD contents in aBaP-packed column and in the effluent before and after ozonation weremeasured to establish a mass balance. In this experiment, the column waspacked with glass beads and 150 mg BaP that amounted to a total demandof 436 mg O₂ as determined by COD test. Ozonated water was elutedthrough the column at 44 mL/min for 4 hours with a total throughput of9.75 L. Influent and effluent O₃ concentrations were frequently measuredat 4.77 mg/L and 0.0 mg/L, respectively. Parent compound in the columnwas measured before and after ozonation, and none was found in theeffluent. The COD contents in the column were measured before and afterozonation, as well as that in the effluent. The results are shown inTable B-III.

The total amount of O₃ consumed in this 4-hr experiment was 0.97 mmol or46.5 mg (i.e., 4.77 mg/L×9.75 L), which would mineralize up to 0.063mmol or 15.9 mg BaP according to this stoichiometric equation:C₂₀H₁₂+15.3O₃=20CO₂+6H₂O. The amount of O₃ consumed could reduce thetotal COD of the system by 15.5 to 46.6 mg of O₂ demand depending on thenumber of oxygen atoms of O₃ involved in the oxidation. The total amountof BaP degraded in this experiment was 32.5 mg or 0.128 mmol. The moleratio of consumed ozone to consumed pyrene was 0.97/0.128=7.6; thus, 7.6moles of ozone were consumed for each mole of BaP degraded. Thisobserved ratio of 7.6 is lower than that of 15.3 theoretically requiredfor the complete mineralization of BaP. Thus, significant amounts ofintermediates and byproducts would be expected either in the effluent oras residuals in the column, which were indeed observed and evidenced bythe higher measured COD due to intermediates in the effluent. The largerequivalents of O₃ consumed per BaP degraded must be recognized with thefact that many observed compounds were products evidently from repeatedattacks by O₃ or its secondary radicals, which inevitably requiredhigher than unit molar equivalent of O₃.

The COD measurements of Table B-III also indicated a reduction of COD inthe system (column residual plus effluent) by 36 mg O₂. This value lieswell within 18 to 53 mg O₂ afforded by O₃ over the experiment duration.This means that the supplied O₃ was primarily consumed in convertingparent BaP to intermediates thereby reducing the system COD, althoughdecomposing of O₃ via hydrolysis was also occurring.

From the viewpoint of applying biological treatment following ozonation,it is desirable to have a lower ratio in consumed ozone to consumed BaPbut higher COD and BOD values in the effluent. Such a system willchemically pre-treat the largely insoluble BaP into dissolvedintermediates that are accessible and biodegradable. Whereas O₃ and itsradical oxidants are capable of mineralizing BaP and its derivatives asin a batch reactor, the pretreatment of BaP with O₃ as in a flow-throughsystem makes effective use of chemical oxidation by generatingintermediates that can be subsequently biodegraded. For otherwisescantly accessible BaP solid, the combined chemical-biological treatmentscheme promotes efficient use of chemical oxidant for pretreatment andviable biodegradation of the resulting nontoxic, water-soluble,biodegradable intermediates.

Example C Degradation and Remediation of Oil Spills

Materials and Methods

The Bipolar Homogenous Solvent System

In this work example, an innovative treatment is developed for spiltoils from southern Kuwait desert that involves ozonation in ahomogeneous solvent system. The solvent is constituted of misciblen-heptane and acetic acid (1:1 by volume), both of that are relativelyinexpensive, environmentally benign, and biodegradable. The solventsystem allows dissolution of oil in its non-polar heptane constituent,while the hydrophilic acetic acid keeps the progressively more polarintermediates and byproducts being formed from ozonation of oil insolution as the reaction continues. The n-heptane and acetic acid werechosen because of their relatively slow reaction rate with ozone(k_(O3)[M⁻¹ s⁻¹] about 10⁻³˜1 for alkanes and 3×10⁻⁵ for acetic acid.

The amount of oil to be employed in the bipolar solution was determinedby its solubility in n-heptane and bipolar solvent. With an addition of2.5 ml of distilled deionized water (about 5%) to a homogenous solution50 ml of n-heptane and 50 ml of acetic acid, the solution was separatedinto two distinct phases: the n-heptane containing PAHs and otherhydrophobic intermediates, and acetic acid phase (95% of acetic acid/5%DD water) containing the hydrophilic byproducts. In this example, about480 mg/L of oil in the bipolar solvent was ozonated for differentdurations and the solution contents were analyzed accordingly. Bothbyproducts in the n-heptane phase and in the 95% acetic acid phase wereanalyzed by GC/FID, GC/FPD, and GC/MS after the separation. Ozonestability in different solvents: bipolar solvent, n-heptane, 95% aceticacid/5% DD water, and pure acetic acid were also tested by stoppingafter 20 minutes of ozonation and monitoring the ozone concentrationimmediately after.

Ozonation Reaction Kinetics in Bipolar Solvent

Ozonations were performed at room temperature (about 20° C.), andreactions were monitored over periods of 15 seconds to 3 hours. Thepseudo-first order rate constants were evaluated by linear regression ofall data according to the equations given below. The reaction of ozonewith contaminants can be expressed as a second order reaction:

−dCA/dt=kCA[O₃]  (1)

where:dCA/dt=rate of reaction of contaminantk=reaction rate constantCA=concentration of contaminant[O₃]=concentration of ozone

In the present of excess and constant ozone concentration, the rate lawfor the reaction can be considered as pseudo-first order:

−dCA/dt=k′CA  (2)

where

−dCA/dt=rate of reaction of contaminantk′=pseudo-first order rate constantCA=concentration of contaminant.

Chemicals

Degraded spilt oil comes from oil lakes in the southern desert ofKuwait. N-Heptane (Fisher Scientific) of HPLC grade and acetic acid(99%, Mallinckrodt) were used as cosolvents in a batch reactor. Stockand working indigo blue solution were prepared from potassium indigotrisulfanate (C₁₆H₇N₂O₁₁S₃K₃, Aldrich Co.) for ozone concentrationmeasurements per Standard Methods. Low-organic (<15 ppb as TOC), low-ion(resistivity>18 M′Ω-cm), and nonpyrogenic (up to 4 log reduction withreverse osmosis pretreatment) distilled-deionized water was used in allprocedures (4-stage MILL-Q Plus system, Millipore Co.). Other chemicalsused in this research were of reagent grade.

Analytical Methods and Equipment

Ozone was generated by an ozone generator (Model T-816, PolymetricsCorp.) from dry and filtered air at an applied voltage of 65V an airflow rate of 2 L/min. The concentration of ozone in the bipolar solventwas determined by absorbance at 270 nm with a spectrophotometer (HP 8452UV-Vis spectrophotometer, Hewlett Packard Co.) using a predeterminedextinction coefficient of 1955 M⁻¹ cm⁻¹. This extinction coefficient wasobtained by correlation with actual ozone concentrations in the bipolarsolvent, which were measured by contacting 10 mL of O₃-saturated bipolarsolvent with 50 mL of standard Indigo Blue solution in a separatoryfunnel, following calibration procedures at 600 nm similar to the IndigoBlue method.

Samples containing oil and intermediates in n-heptane, 95% acetic acid,and bipolar solvent were analyzed respectively using a gas chromatograph(GC). GC/FID analyses were carried out using a HP 5890 (Hewlett PackardCo.) fitted with a capillary column (DB-1 non-polar column, 60 m×0.25mm×0.25 um, J&W Co.) and a flame ionization detector (FID). The GC/FIDwas interfaced and programmed with the HP Chemstation software (HewlettPackard Co.) A 5:1 split and 1 uL sample injection were used. Thechromatographic oven was held at 35° C. for 1 min then linearlyincreased at 5° C. per min to 300° C. with a 30 minutes hold.

Samples were analyzed with a GC (HP 6890) with a capillary column (DB-1non-polar column, 60 m×0.25 mm×0.25 um, J&W Co.) interfaced to a massspectrometry detector (MS) (HP6800) and programmed with the HPChemstation software (Hewlett Packard Co.). A split ratio of 5:1,solvent delay at 10 minutes, and scan range from m/z 15 to m/z 550 at1.4 scan/sec were used. The oven temperature was held at 35° C. for 1min then linearly increased at 5° C. per min to 300° C. and held for 30minutes. The HP Chemstation library (Hewlett Packard Co.) was used fortentative identification of peaks as a supplement to mass spectral andretention time characteristics. In addition, comparison of parentcompound structure and interpretation of mass spectra of theintermediates from ion fragmentation were performed particularly for theidentification of key intermediates.

Samples were also analyzed with a GC/FPD (HP 5890) for bulkcharacterization of sulfur-containing compounds. GC/FPD analyses werecarried out using a HP 5890 (Hewlett Packard Co.) fitted with acapillary column (DB-1 non-polar column, 60 m×0.25 mm×0.25 um, J&W Co).The GC/FPD was interfaced and programmed with the HP Chemstationsoftware (Hewlett Packard Co.) A 10:1 split and 1 uL sample injectionwere used. The chromatographic oven was held at 35° C. for 2 min thenlinearly increased at 4° C. per minute to 225° C. and continuingincreased at 8° C. per minute to 300° C. with a 40 minutes hold.

Batch Reactor

A glass batch reactor with a working volume of 300 mL was used. Mixingin the reactor was provided by a magnetic mixer operating at 250 rpm.After preparing about 50 ml of 960 mg of oil per liter of n-heptane, 50ml of acetic acid was added into the reactor (resulting in a 480 mg/Lsolution of oil in the bipolar solvent). Ozone was sparged into thereactor near the bottom through a glass dispersion tube (ACE glass Inc.)Reaction batches were stopped after 0.25, 0.5, 1, 2, 3, 4, 5, 10, 20,40, 60, 120, and 180 minutes of ozonation. Residual ozone was removedfrom solution by purging with a gentle N₂ stream for 1 min. Samples werekept in 2-mL vials and preserved at 5° C. if necessary prior to GCanalysis. Qualitative and quantitative analyses of oil and oxidized oilwere performed simultaneously. All samples were concentrated by a gentlestream of N₂ gas to best retain the intermediates with lower molecularweights.

Results and Discussion

The oxidative degradation of different fractions of hydrocarbons byozonation in the bipolar solvent, the reactivity of aromatic sulfurcompounds in ozonated environment, and the biodegradability of ozonatedoil will be discussed. All major compounds discussed are illustrated inTable C-1.

Ozonation of Saturate Factions of Spilt Oil

The saturate factions are normal, iso-paraffin (branched alkanes) andcyclic alkanes of the spilt oil. The n-icosane (about 6% estimated wt%), pristane and phytane are chosen to represent normal alkanes andbranched alkanes respectably. The ozonation results of these threecompounds in the bipolar solvents are shown in FIG. 16. The n-icosanewas shown more resistant to ozone than pristane and phytane. Thetentative estimated pseudo-first order rate constants (as shown in TableC-2) for n-icosane, pristane, and phytane in the complex mixture are1.98×10⁻³, 1.30×10⁻², and 1.78×10⁻² s⁻¹ respectively.

Cyclic alkanes with long alkylated chain were also degraded by ozonationas depicted in FIG. 16. Steranes and terpanes with four cyclic saturatedrings and hopanes with five cyclic saturated rings are detected by GC/MS(about 3.0% estimated wt %). Alkyl steranes were depleted throughozonation much faster than alkyl hopanes and terpanes. The reason tochoose steranes, terpanes, and hopanes to represent saturated cycliccompounds is that they are not only the common constituents in the crudeoil but also very resistant to biodegradation. The length of thealkylated chain does not have significant influence on the rate ofreaction with ozone in this example. The degradation of n-icosane,pristane, phytane, steranes, and hopanes during the ozonation areillustrated in FIG. 16 and the tentative estimated pseudo-first orderrate constants are shown in Table C-2. It is noticed that saturatedcyclics (k′=2.57×10⁻¹ s⁻¹) undergo much faster ozonation than branchedalkanes (k′=1.54×10⁻² s⁻¹); and normal alkanes (k′=1.98×10⁻³ s⁻¹) werelast readily oxidized by ozone. Small amounts of the normal alkanes willlikely degraded into alkenes, alcohols, ketones, and esters during 3hours of ozonation. They observed no substantial change in the percentamount of n- and iso-paraffin carbons, but a noticeable increase innaphthenic carbon (cyclic alkanes). They explained that the formation ofsuch saturated structures could be due to possible condensation reactioncatalyzed by Cu metal in metal catalyzed oxidation processes.

Ozonation of Aromatic Factions of Spilt Oil

The bipolar solvent system consists of both non-polar heptane andhydrophilic acetic acid. The heptane component enables high solubilityof PAHs and the acetic acid keeps the polar intermediates and byproducts in solution. The bipolar solvent system maintains effectiveexposure of all compounds to ozone throughout the course of reaction andprevents the formation of sludge residues. The aromatic factions ofspilt oil discussed in this example are polycyclic aromatichydrocarbons, such as alkylnaphthalenes, alkylphenanthrenes, andalkylbenzenes; and sulfur heterocycles such as alkylbenzothiophines(ABTs), alkyldibenzothiophines (ADBTs), and alkylbenzonaphthothiophines(ABNTs).

The naphthalenes, alkylnaphthalenes, phenanthrenes, andalkylphenanthrenes ozonated in the bipolar solvent are presented in FIG.17 a. All of the naphthalenes and phenanthrenes destroyed by ozonewithin 30 seconds. The number of the aromatic rings does affect the rateof reaction of ozone electrophilic attacks. In general, thephenanthrenes with 3 aromatic rings, lower aromaticity, and with higherelectron density will likely more reactive with electrophilic ozone thannaphthalenes. As shown in Table C-2, phenanthrenes (k′=1.12×10² s⁻¹) aredepleted faster than naphthalenes (k′=1.11×10¹ s⁻¹). The ring cleavagesof PAHs on the bond and/or atoms with lowest localization energy byozone molecule in the bipolar solvent has been observed in Examples Aand B for pyrene and benzo[a]pyrene. The more hydrophilic intermediatesand byproducts, such as aldehydes, ketones, lactones, and carboxylicacids have been generated during the ozonation. Oxygenated biphenyls,one type of the significant intermediates in ozonation of 3 or morerings' aromatics has been observed in the system after the degradationof phenathrenes as presented in FIG. 17 b. Both diphenic acid andcarbonyl oxide was primary intermediates from ozonated phenanthrene inthe non-participating solvent. The long alkyl chain based alkylbenzenes(less than 2.0% estimated wt %) have also been degraded in the bipolarsolvent via ozonation (as see in FIG. 18). The long alkyl chain with 17to 21 carbons on the alkylbenzenes (k′=1.75×10⁻¹ s⁻¹) made them muchlike normal alkanes being relatively more resistant to ozone thannaphthalenes and phenanthrenes.

Table C-2 present the tentative estimated rate of degradation of all thesaturate compounds and aromatic compounds in spilt oil during theozonation in the bipolar solvent system. Naphthalenes, phenanthrenes,benzothiophines, and benzonaphthothiophines were very oxidized in thebipolar solvent. Dibenzothiophines, alkylbenzenes, steranes, and hopaneswere substantially stay in solvent in first five minutes, and thendegraded rapidly. N-icosane, pristane, and phytane were relativelyresistant to ozonation. In this example, both saturated and aromatichydrocarbons in the oil decreased via ozonation result in the formationof oxygenates (polar compounds). The aldehyde, ketone, lactone,carboxylic acid, alkene, alcohol, ketone, and ester type ofintermediates and byproducts have been generated during the ozonationand observed in GC/MS.

These compounds usually are more hydrophilic and mainly sludgeprecursors in single organic solvent systems. However, all hydrophobicand hydrophilic compounds/intermediates will stay in dissolved form tofurther ozonation in the bipolar solvent. The results from FIG. 21 andTable C-2 indicated that polycyclic aromatic compounds in the spilt oilundergo more oxidative degradation than saturated compounds. Thepreferential degradation order of ozonation in the bipolar solvent onthe oil constituents was observed as follow: the aromatics (i.e. PAHsand thiophines)>cyclic alkanes (i.e. steranes, terpanes, andhopanes)>branch alkanes (i.e. pristane and phytane)>n-alkanes (i.e.icosane). This result suggested that ozonation as a pretreatment forspilt oils can preferentially eliminate or convert recalcitrantfractions such as aromatics and aromatic sulfur into more bioavailableand water-soluble compounds to be with saturated fraction of oil forsubsequent biological degradation.

Ozonation of Aromatic Sulfur Compounds

After carbon and hydrogen, sulfur is typically the third most abundantelement in petroleum, ranging from 0.05 to 5% w/w in crude oil. Thesulfur-containing compounds in the petroleum are responsible for the airpollution caused by diesel exhaust gas (SO_(x)). Thiophine compounds arealso found in some waste streams particularly in wastewater from oilrefineries. Condensed thiophines comprise a significant portion of theorganosulfur compounds in petroleum. Thiophenes, the model compounds inbiodegradation studies, are refractory polycyclic aromatic sulfurcompounds present in coal and crude oil. The alkyl-substituteddibenzothiophines were reported as the most recalcitrant tobiodegradation within the aromatic fraction of petroleum.

From FIGS. 19 a and 19 c, the benzothiophines and benzonaphthothiophineswere completely eliminated by ozonation in the bipolar solvent systemswithin 1-minute. However, dibenzothiophines stay in bipolar solvent forabout two hours of ozonation (in FIG. 19 b). In the reaction withelectrophiles such as ozone under non-participated solvent, attack oncarbon of thiophines is the predominant mode of reaction rather thanreaction on sulfur. This feature suggested the ozonation of these threesulfur contained compounds will be similar to ozonation of aromatics.The 3 aromatic rings fused benzonaphthothiophines with less aromaticity,higher frontier electron density, and lower localization energy on atomsand bonds will undergo more rapid reaction with ozone. In comparisonwith ozonation of benzothiophines and dibenzothiophines, the reactionrates of these were separated by position order of reactivity viaelectrophiles. The position order of reactivity on benzothiophine wasdetermined to be 3>2>6>5>4>7, which indicated sulfur containing ring aremore reactive to electrophiles than benzene. The electrophilicsubstitutions on dibenzothiophines occur predominantly at the positionpara- to the sulfur atom such as position −2 and −8, which is mainly onboth fused benzenes. Since the benzene rings are less readily attackedby ozone than sulfur-fused rings, benzothiophines with exposed sulfurfused ring undergo much severe oxidation than dibenzothiophines.

Even the catalytic hydrodesulfurization (HDS) method employed in therefining processes has difficulty in the desulfurization ofdibenzothiophine and its derivatives among sulfur-containing compoundsin the light oil. The ozonated thiophines in non-participating solventas bipolar solvent likely will form o-hydroxybenzene sulfonic acid,o-sulfobenzoic acid, and homophthalic acid. The mass spectra of ozonatedthiophines showed fragments of m/z 60 and 73 corresponding to theunderivatized acids.

Bulk characterization of sulfur-containing compounds in GC/FPD caneffectively separate the low molecular weight organic sulfur compoundsthat mainly are organic polysulfides from the high molecular weightorganic sulfur compounds that mainly represent high molecular weightresins (more than six condensed-ring aromatics with heteroatoms contentsand polar compounds) and asphaltenes (the mixture ofpolydispersed-condensed polyaromatic units, with heteroatoms contents,bearing alicyclic sites, and substituted and connected with each othervia aliphatic chains) besides the alkylated thiophines,dibenzothiophines, and benzonaphthothiophines. FIG. 20 monitored the lowmolecular weight sulfur compounds cumulated along with ozonation, andthe high molecular weight sulfur compounds are gradually decrease withozonation. This result from FIGS. 18 and 19 showed that the highmolecular weight OSC were mineralized into low molecular weight OSC byozonation in the bipolar solvent.

Biodegradability of Ozonated Oil

As a potent treatment agent, O₃ tends to self-decompose in aqueousenvironment or react with organic solvents. FIG. 23 compares thesolubility and stability of O₃ in aqueous and organic solvents ofdifferent compositions under the employed experimental conditions andequipment settings. Ozone exhibits higher solubility and stability inthe bipolar solvent used in the present example than in water or heptanealone. The stability of the solvent system itself subject to ozonationwas tested by conducting ozonation experiments of the solvent systemwith and without contaminants.

Scenarios of remediation application will likely call for continualozonation of spilt oil (or other wastes) at higher concentrations, whichwill more likely result in an abundant formation of intermediates. Toassess the level of ozonation pretreatment that would be required torender aromatic compounds in spilt oil and their daughter compoundsco-metabolizable, the inhibitory effect of the intermediates fromozonation of spilt oil on biological treatment were studied. Aftervarying duration of ozonation in the bipolar solvent as describedpreviously, the separated 95% acetic acid solutions laden withintermediates were diluted to 5% acetic acid solution and were testedfor E-coli toxicity as well as BOD₅.

FIG. 22 a shows toxicity of the intermediates and FIG. 22 b the BOD₅according to ozonation duration. As shown, the E-coli toxicity of theintermediates increased from a +5.30% initially to +17.65% after 10-minozonation then decreased to −4.16% after 60-min ozonation and remainedrelatively stable and nontoxic thereafter. It should be noted for theE-coli test the nontoxic range is within ±10%, and the toxic rangeoutside which. The reason of nontoxic results initially mainly due tohydrophobic nature of spilt oil, which prefer to stay with non-polarheptane and limited compounds from oil dissolved into acetic acid. Afterozonation, polar intermediates formed and readily dissolved into aceticacid, which contributed to the toxicity and BOD₅.

The BOD₅ results show a gradual increase from little registered BOD₅ to245 mg/L throughout the course of ozonation except initial BOD₅ with 230mg/L, whereas theoretical calculation of the ultimate BOD due to aceticacid present in the sample amounts to 320 mg/L. In other words, the5-day BOD (BOD₅) of 230 mg/L measured for the intermediates-ladenozonated sample constituted 72% of the ultimate BOD. This ratio of BOD₅to ultimate BOD is not uncommon for readily biodegradable substances,and it indicates that the degradation of acetic acid has not beeninhibited by the presence of the intermediates. The reason of 230 mg/Lof initial BOD₅ was mostly due to hydrophobic nature of spilt oil, whichprefer to stay with non-polar heptane and limited compounds from oildissolved into acetic acid. In other word, 230 mg/L measured BOD₅ wereprimarily from 5% acetic acid. The 245 mg/L of BOD5 after 3-hoursozonation, 15 mg/L of the BOD₅ should be contributed from those polarintermediates.

The toxicity and BOD results are consistent with each other, suggestingthat for the initial spilt oil loading of 480 mg/L, 60 min of ozonationin the bipolar system is sufficient to render it nontoxic,co-metabolizable to the E-coli bacteria. From a viewpoint of overallprocess efficiency, the results indicate viability of the bipolarsolvent system in incorporating a sequential chemical-biologicaltreatment scheme.

Conclusions

In this example, the homogeneous phase of the bipolar solvent makes thetarget compounds constantly susceptible to attack and degradation at themolecular level. All different fractions of constituents in thepetroleum were able to react with ozone homogeneously in the bipolarsolvent. The preferential degradation order of ozonation in the bipolarsolvent on the oil constituents was also observed: the aromatics (i.e.PAHs and thiophines)>cyclic alkanes (i.e. steranes, terpanes, andhopanes)>branch alkanes (i.e. pristane and phytane)>n-alkanes (i.e.icosane). The toxicity and BOD results suggested that for the initialspilt oil loading of 480 mg/L, 60 min of ozonation in the bipolar systemis sufficient to render it nontoxic, co-metabolizable to the E-colibacteria. Therefore, the ozonation of spilt oil in the bipolar solventcan eliminate and transform the toxic and recalcitrant aromatichydrocarbons which will undergo more oxidative degradation thansaturated compounds into acetic acid with biodegradable saturatedmolecules for the subsequent biotreatment or reozonation.

In separate experiments of spilt oil employing heptane as the solesolvent, precipitates occurred shortly after ozonation commenced.Separation of the heptane solution from the solid precipitate andsubsequent GC analyses of the solution and the solid revealed only theparent nonpolar compounds remained in solution. While a nonpolar solventsuch as heptane dissolves spilt oil and makes it readily susceptible toO₃ attack and degradation, the nonpolar solvent fails to retain thepolar intermediates in solution, resulting in the formation of a solidprecipitate shortly after ozonation begins. Therefore, the use of thebipolar solvent eliminates the occurrence of a solid phase that oftenbecomes the rate-limiting step in the waste treatment sequence. Thebipolar solvent system such as the heptane/acetic acid (1:1, v/v) can beuseful in tackling recalcitrant compounds such as pyrene,benzo[a]pyrene, and other heavy polycyclic aromatic hydrocarbons. Thesecompounds are recalcitrant partly due to their hydrophobic nature thatrenders them highly insoluble, thus rendering inaccessible to microbesand even to chemical oxidant such as O₃ in the aqueous phase.

The bipolar solvent as described is readily separated into two phases.By adding a small amount of water, the two phases separate allowing theheptane devoid of the contaminant to be reused for another treatmentcycle and the acetic acid now laden with biodegradable intermediates(including acetic acid itself) amenable to further biologicaldegradation.

While this invention has been described with reference to certainspecific embodiments and examples, it will be recognized by thoseskilled in the art that many variations are possible without departingfrom the scope and spirit of this invention, and that the invention, asdescribed by the claims, is intended to cover all changes andmodifications of the invention which do not depart from the spirit ofthe invention.

TABLE A-I BOD and COD of pyrene solutions (means ± standard deviationsof triplicates). BOD₅ COD BOD₅/COD Solution (mg/L) (mg/L) ratio (#)Pyrene 0.83 ± 0.15  1.0 ± #0.01 0.83 (saturated, not ozonated) Batch(ozonated, 10 min) 1.75 ± 0.65 2.67 ± 1.2  0.66 Column effluent 7.30 ±0.20 13.7 ± 0.76 0.53 (ozonated, composite)

TABLE A-II Intermediates and products in the ozonated column effluent,as identified by GC/MS. (• found; ◯ not found) Retention Ozonatedeffluent at interval Rinse Time before 90- 120 (min) Compound ozonation15-30 min 30-60 min 60-90 min 120 min min after 14.6 unknown (m/z 154)(26) • • • • • • 15.3 unknown (m/z 139) (27) • • • • • • 20.2 ethanol,2-[2-butoxyethoxy]- (14) • ◯ ◯ ◯ ◯ ◯ 22.5 phthalic anhydride (7) • • • •◯ • 23.2 propanoic acid, 2-methyl-, butyl ester (15) • • • • ◯ • 25.3tetradecane (17) • • • • ◯ • 28.1 pentadecane (18) • • ◯ ◯ ◯ ◯ 28.2butylated hydroxytoluene (11) • • ◯ ◯ ◯ • 29.5 diethyl phthalate (6) • ◯◯ ◯ ◯ ◯ 30.7 hexadecane (19) • • • ◯ ◯ ◯ 33.0 nonyl phenol (13) • • • ◯◯ • 37.6 4H-cyclopenta[def]phenanthrene (8) • • • • • • 37.9 dibutylphthalate (12) • • • • • • 38.3 hexadecanoic acid (16) • • • ◯ ◯ • 38.8xanthone (10) ◯ ◯ • • • ◯ 41.6 henicosane (21) • • • • • • 41.7 pyrene(1) • • • • • • 43.3 2,2′,6,6′-biphenyltetraaldehyde (3) ◯ • • • • •43.5 docosane (22) • • • • • • 45.1 tricosane (23) • • • • • • 45.3Benzylbutyl phthalate (5) • • • • • • 46.8 4,5-phenanthrenedialdehyde(2) ◯ • • • • • 46.9 tetracosane (24) • • • • • • 48.4Cyclopenta[def]phenanthrene (9) • • • • • • 48.7 1,2-benzenedicarboxylicacid, diisooctyl (4) • • • • • • 48.8 dibutyl phthalate (12) • • • • • •48.9 pentacosane (20) • • • • • • 50.4 hexacosane (28) • • • • • • 53.66-propyl tridecane (25) • • • • • •

TABLE A-III Intermediates and products in the ozonated effluent aftervarious degree biotreatment, as identified by GC/MS. (• found; ◯ notfound) Retention Time Biological Incubation of Ozonated Effluent (min)Compound 0 day 5 days 10 days 15 days 20 days 14.6 unknown(m/z 154) (26)• • • • • 15.3 unknown(m/z 139) (22) • • • • • 20.2 ethanol,2-[2-butoxyethoxy]- (14) ◯ ◯ ◯ ◯ ◯ 22.5 phthalic anhydride (2) • ◯ ◯ ◯ ◯23.3 propanoic acid, 2-methyl-, butyl ester (15) • ◯ ◯ ◯ ◯ 25.3tetradecane (17) • • • • • 28.1 pentadecane (18) ◯ ◯ ◯ ◯ ◯ 28.2butylated hydroxytoluene (11) • • • • • 29.5 diethyl phthalate (6) ◯ ◯ ◯◯ ◯ 30.7 hexadecane (19) • ◯ ◯ ◯ ◯ 31.0 phosphoric acid tributyl ester(31) ◯ ◯ ◯ • • 33.0 nonyl phenol (13) • ◯ ◯ ◯ ◯ 36.8 cyclododecane (29)• • • • • 37.6 4H-cyclopenta[def]phenanthrene (8) • ◯ ◯ ◯ ◯ 37.9 dibutylphthalate (12) • • • • • 38.3 hexadecanoic acid (16) • ◯ ◯ ◯ ◯ 38.8xanthone (10) • ◯ ◯ ◯ ◯ 41.6 henicosane (21) • ◯ ◯ ◯ ◯ 41.7 pyrene (1) •◯ ◯ ◯ ◯ 43.0 biological culture (m/z 226) (30) • • • • • 43.32,2′,6,6′-biphenyltetraaldehyde (3) • ◯ ◯ ◯ ◯ 43.5 docosane (22) • ◯ ◯ ◯◯ 45.1 tricosane (23) • • • • • 45.3 benzylbutyl phthalate (5) • • • • •46.8 4,5-phenanthrenedialdehyde (2) • • • • • 46.9 tetracosane (24) • •• • • 48.4 cyclopenta[def]phenanthrene (9) • • • • • 48.71,2-benzenedicarboxylic acid, • • • • • diisooctyl (4) • • • • • 48.8dibutyl phthalate (12) • • • • • 48.9 pentacosane (20) • • • • • 50.4hexacosane (28) • • • • • 53.6 6-propyl tridecane (25) • • • • •

TABLE A-IV Mass balance for the pyrene-loaded column before and afterozone treatment (one typical run). Column Column Effluent Amount reducedInitial loading after ozonation column and efflu

Pyrene (mg)^(a) 147.2 74.6 1.24 147.2 + 1.24 − 74

 = 73.8 (50%) 4,5-Phenanthrenedialdehyde (mg)^(a) 0 0 1.432,2′,6,6′-Biphenyltetraaldehyde (mg)^(a) 0 0 1.72 COD (mg O₂)^(b) 432280 116 432 − 116 − 280 = 36 ^(a)Amounts of substance (mg) in the columnas quantified by GC/FID. ^(b)Oxygen demand (mg O₂) in the column asdetermined by COD test.

indicates data missing or illegible when filed

TABLE B-I BOD and COD of benzo[a]pyrene solutions (mean ± standarddeviation of triplicates). BOD₅ COD BOD₅/COD Solution (mg/L) (mg/L)ratio (#) Benzo[a]pyrene — 0.3 ± 0.5 — (saturated, not ozonated) Batch(ozonated, 50 min) — 12.7 ± 1.5  — Column effluent 4.2 ± 0.20 13.7 ±0.76 0.43 (ozonated, composite of 4 h)

TABLE B-II Intermediates and products from ozonation of benzo[a]pyrene,as Identified by GC/MS. Compound Retention time (min) m/z Compound name1 9.83 168 No ID 2 9.89 112 1-Pentene, 2-isopropyl 3 9.95 140 1-Hexene,4-methyl, 2-isopropyl 4 10.25 140 1-Pentene, 3,4-dimethyl, 2-propyl 510.46 140 1-Pentene, 3,4,4-trimethyl, 2-ethyl 6 10.93 140 1-Pentene,3,4-methyl, 2-dimethyl 7 11.01 140 1-Hexene, 3,4-dimethyl, 2-ethyl 811.10 140 No ID 9 11.21 140 1-Hexene, 4,5-dimethyl, 2-ethyl 10 11.27 1401-Hexene, 4,5-dimethyl, 2-ethyl 11 11.33 140 1-Pentene, 3-methyl,2-isobutyl 12 11.80 140 1-Hexene, 4,5-dimethyl, 2-ethyl 13 11.88 1402-Hexene, 2,3,4,5-tetramethyl 14 12.02 140 1-Hexene, 4,5-dimethyl,3-ethyl 15 12.51 154 No ID 16 13.37 154 3-Octane, 5-methyl, 3-ethyl 1713.98 154 3-Nonene, 6,8-dimethyl 18 14.17 154 1-Hexene, 3,5-dimethyl,2-iospropyl 19 14.34 154 3-Nonene, 6,8-dimethyl 20 14.96 154 13-octene,25,7-trimethyl 21 15.48 154 3-Decene, 9-methyl 22 15.66 139 Butanoicvinyl anhydride 23 16.50 123 No ID 24 17.05 334 Decafluorobiphenyl(Internal standard) 25 17.42 182 No ID 26 24.16 429 No ID 27 36.81 2423-methylchrysene 28 37.60 272 7-methyl-8-propanalpyrene 29 38.42 149Phthalic anhydride 30 38.70 272 4-Methyl-5-methanal-chrysene 31 45.66127 Pentadecane (C15) 32 47.38 149 Benzole acid, ethyl ester 33 47.75163 Benzole acid, propyl ester 34 47.92 141 Tridecane (C16) 35 49.04 163Bis (2-ethylhexyl) phthalate 36 49.27 293 1,2-Benzenedicarboxylic acid,diisononyl ester 37 52.16 293 1,2-Benzenedicarboxylic acid, diisononylester 38 52.21 293 1,2-Benzenedicarboxylic acid, diisononyl ester 3952.37 293 1,2-Benzenedicarboxylic acid, diisononyl ester 40 52.48 2931,2-Benzenedicarboxylic acid, diisononyl ester 41 52.57 2931,2-Benzenedicarboxylic acid, diisononyl ester 42 52.68 2931,2-Benzenedicarboxylic acid, diisononyl ester 43 52.78 2931,2-Benzenedicarboxylic acid, diisononyl ester 44 52.92 2931,2-Benzenedicarboxylic acid, diisononyl ester 45 53.10 2931,2-Benzenedicarboxylic acid, diisononyl ester 46 53.20 2931,2-Benzenedicarboxylic acid, diisononyl ester 47 53.25 2931,2-Benzenedicarboxylic acid, diisononyl ester 48 53.51 2931,2-Benzenedicarboxylic acid, diisononyl ester 49 53.65 2931,2-Benzenedicarboxylic acid, diisononyl ester 50 53.72 2671,2-Benzenedicarboxylic acid, diisononyl ester 51 53.90 2931,2-Benzenedicarboxylic acid, diisononyl ester 52 54.00 2931,2-Benzenedicarboxylic acid, diisononyl ester 53 54.12 2931,2-Benzenedicarboxylic acid, diisononyl ester 54 54.42 2931,2-Benzenedicarboxylic acid, diisononyl ester 55 54.73 2931,2-Benzenedicarboxylic acid, diisononyl ester 56 55.21 2931,2-Benzenedicarboxylic acid, diisononyl ester 57 55.74 2931,2-Benzenedicarboxylic acid, diisononyl ester 58 57.82 197 Octadecane(C18) 59 60.19 197 Octadecane, methyl (C19) 60 62.98 197 nonadecane(C19)61 66.30 197 heneicosane (C21)

TABLE B-III Mass balance for the benzo[a]pyrene-loaded column before andafter ozone treatment (one typical run). Column from Initial loadingEffluent column and After reduced effluent Column ozonation AmountBenzo[a]pyrene 150 117 0.0 150 − 11

 = (mg)^(a) 33.0 (22.0%) COD(mg O₂)^(b) 436 340 78.0 436 − 78

 = 340 36.0 ^(a)Amounts of substance (mg) in the column as quantified byGC/FID. ^(b)Oxygen demand (mg O₂) in the column as determined by CODtest.

indicates data missing or illegible when filed

TABLE C-1 Represent compounds and their structures Represent RepresentType Compounds Structure I Icosane H₃C—(CH₂)₁₈—CH₃ II Pristane

Phytane

III Steranes

R = C₁, C₂, C₃ Tetracyclic Terpane

Pentacyclic Terpanes

R = C₁, C₂, C₃ IV Alkyl Benzenesol

R = C₁₇ to C₂₁; R′ = C₀, C₁ Naphthalenes

R = C₀, C₁, C₂, C₃ Phenanthrenes

R = C₀, C₁, C₂, C₃ V Benzothiophenes

R = C₀, C₁, C₂, C₃, C₄ Dibenzothiophenes

R = C₀, C₁, C₂, C₃ Benzonaphthothiopheness

R = C₀, C₁, C₂, C₃ Alkyl group R = C_(n)H_(2n+1)

TABLE C-2 Tentative estimated pseudo-first-order rate constants underozonated bipolar system Compounds k′ (s⁻¹) nC20 1.98E−03 Pristane1.30E−02 Phytane 1.78E−02 Sum of Steranes 4.94E−01 Sum of Hopanes1.20E−01 Sum of Alkyl Benzenes 1.75E−01 Sum of Naphthalenes 1.08E+02 Sumof Phenanthrenes 1.12E+02 Sum of Benzothiophines 1.57E+02 Sum ofDibenzothiophines 1.96E−01 Sum of Benzonaphthothiophines 6.72E+01Straight Chain (Type I) 1.98E−03 Brainched Chain (Type II) 1.54E−02Saturated Cyclics (Type III) 2.57E−01 Aromatics (Type IV)^(#) 1.10E+02Aromatic Sulfur (Type V) 6.82E+01 ^(#)Excluding long chain alkylbenzenes

1. A method for treating polycyclic aromatic hydrocarbons, the methodcomprising: contacting the polycyclic aromatic hydrocarbons with areaction medium to form a mixture of intermediates and byproducts, saidreaction medium including ozone and a liquid solvent.
 2. The method ofclaim 1, wherein the liquid solvent comprises a non-polar solvent. 3.The method of claim 2, further comprising: using the non-polar solventto solubilize the polycyclic aromatic hydrocarbons.
 4. The method ofclaim 3, wherein the non-polar solvent comprises a hydrocarbon with atleast 7 carbon atoms.
 5. The method of claim 3, wherein the liquidsolvent comprises a polar solvent that is soluble with water and ispresent in an amount and of a sufficiently non-polar character to form asingle phase with the non-polar solvent.
 6. The method of claim 1,wherein the liquid solvent comprises a member selected from the groupconsisting of heptane, acetic acid, and combinations or mixturesthereof.
 7. The method of claim 4, further comprising mixing thereaction medium with sufficient water to form two phases, a non-polarand a polar phase, the non-polar phase comprising the non-polar solventand the polar phase comprising the polar solvent and oxygenatedintermediates.
 8. A method for the degradation of polycyclic aromatichydrocarbon compounds, the method comprising: contacting the polycyclicaromatic hydrocarbon compounds with ozone dissolved in a bipolarsolvent, the bipolar solvent comprising a non-polar solvent and a polarsolvent, the bipolar solvent having solubility to polycyclic aromatichydrocarbon compounds that are insoluble in water as well as tooxygenated intermediates and reaction products of polycyclic aromatichydrocarbon compounds and ozone, and the contacting being for asufficient duration to solubilize and react the polycyclic aromatichydrocarbon compounds with the ozone to form oxygenated intermediates.9. The method of claim 8, wherein the non-polar solvent comprises ahydrocarbon with at least 7 carbon atoms.
 10. The method of claim 8,wherein the polar solvent comprises an organic acid.
 11. The method ofclaim 8, wherein the bipolar solvent comprises heptane and acetic acid.12. The method of claim 8, further comprising mixing the bipolar solventwith sufficient water to form two phases, a non-polar phase comprisingthe non-polar solvent and a polar phase comprising the polar solvent andthe oxygenated intermediates.
 13. The method of claim 8, furthercomprising: separating the polar phase from the non-polar phase;diluting the polar phase with water in an amount to allow microbegrowth; and incubating the polar phase in the presence of microbes tobiodegrade the oxygenated intermediates.
 14. The method of claim 13,wherein the diluted polar phase is biodegraded for sufficient durationto essentially mineralize the oxygenated intermediates.
 15. A method oftreating polycyclic aromatic hydrocarbons, the method comprising:contacting the polycyclic aromatic hydrocarbons with a non-polarsolvent; adding a polar solvent to the non-polar solvent to form asingle miscible phase; and adding ozone to react with the polycyclicaromatic hydrocarbons to form oxygenated intermediates.
 16. The methodof claim 15, further comprising adding water to separate the misciblephase into a polar phase and a non-polar phase, wherein the polar phasecomprises the oxygenated intermediates.
 17. The method of claim 15,further comprising incubating the polar phase in the presence ofmicrobes for a sufficient duration to essentially mineralize theoxygenated intermediates.
 18. The method of claim 15, wherein the polarsolvent comprises an organic acid.
 19. The method of claim 15, whereinthe non-polar solvent comprises a hydrocarbon with at least 7 carbonatoms.
 20. The method of claim 15, wherein at ambient temperature thepolycyclic aromatic hydrocarbons are in a solid phase before beingcontacted with the non-polar solvent.
 21. The method of claim 15,wherein the polycyclic aromatic hydrocarbons are in a liquid phasewithin the single miscible phase.